Mammalian Genome (2018) 29:558–576 https://doi.org/10.1007/s00335-018-9749-4

Enterobacteria and host resistance to infection Eugene Kang1,2 · Alanna Crouse2,3 · Lucie Chevallier4,5 · Stéphanie M. Pontier6 · Ashwag Alzahrani6 · Navoun Silué6 · François‑Xavier Campbell‑Valois6,7   · Xavier Montagutelli4   · Samantha Gruenheid1,2   · Danielle Malo2,3,8  Received: 16 February 2018 / Accepted: 14 May 2018 / Published online: 21 May 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Enterobacteriaceae are a large family of Gram-negative, non-spore-forming bacteria. Although many species exist as part of the natural flora of animals including humans, some members are associated with both intestinal and extraintestinal diseases. In this review, we focus on members of this family that have important roles in human disease: Salmonella, Escherichia, Shigella, and Yersinia, providing a brief overview of the disease caused by these bacteria, highlighting the contribution of animal models to our understanding of their pathogenesis and of host genetic determinants involved in susceptibility or resistance to infection.

Introduction Enterobacteriaceae are a large family of Gram-negative, non-spore-forming bacilli consisting of over 50 genera and 210 species (Jenkins et al. 2017). The members of this family are ubiquitous and are distributed across diverse ecological niches in both terrestrial and aquatic environments including the soil, water, plants, and animals (Jenkins et al. 2017). Although many species exist as part of the natural flora of animals including humans, Enterobacteriaceae are frequently associated with both intestinal and extraintestinal diseases (Donnenberg 2015). The following members are

Eugene Kang, Alanna Crouse, Lucie Chevallier, and Stéphanie M. Pontier have contributed equally to this work. François-Xavier Campbell-Valois, Xavier Montagutelli, Samantha Gruenheid, and Danielle Malo have contributed equally to this work. * Danielle Malo [email protected] 1

important pathogens in the setting of public health and are the primary focus of this review: Salmonella, Escherichia, Shigella, and Yersinia. Together, these pathogens are a significant cause of morbidity and mortality worldwide, and the emergence of multidrug resistance poses important challenges for the control and prevention of Enterobacteriaceae infections. Animal models have long been used to address a number of scientific questions that have led to major breakthroughs in basic biological science and medical research. Animal models allow for the study of the virulence factors and mechanisms of pathogenic bacteria that contribute to disease and are hence a valuable tool in the context of infectious disease. Human resistance to infectious disease is a complex trait with varying modes of inheritance. Monogenic inheritance, in which a phenotype is governed by a single gene/locus, is seen in cases of rare Mendelian diseases 5



Mouse Genetics Laboratory, Department of Genomes and Genetics, Institut Pasteur, Paris, France

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Department of Chemistry and Biomolecular Sciences, Centre for Chemical and Synthetic Biology, University of Ottawa, Ottawa, ON, Canada



Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada

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McGill Research Center on Complex Traits, McGill University, Montreal, QC, Canada

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Department of Human Genetics, McGill University, Montreal, QC, Canada

Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada

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4

U955 - IMRB, Team 10 - Biology of the neuromuscular system, Inserm, École Nationale Vétérinaire d’Alfort, UPEC, Maisons‑Alfort, France

Department of Medicine, McGill University, Montreal, QC, Canada



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such as primary immunodeficiencies (reviewed in Alcais et al. 2009). In contrast, polygenic inheritance involves many genes making smaller contributions to produce the observed phenotype. The “major gene/locus” effect concept is a compromise between monogenic and polygenic inheritance, which involves a single or few genes of lower penetrance having the largest effect with smaller contributions made by additional genes and/or environmental factors (Casanova and Abel 2007). Given the complexity of infectious diseases at the genetic level, a wide range of tools has been needed to explore this field. The ability to manipulate the genomes in animal models has been an asset to our current understanding of host–pathogen interactions with both reverse and forward genetic approaches being of use. Reverse genetics is a gene-driven approach that determines gene function by altering either gene sequence or expression and then studying the resulting phenotype. Methods of reverse genetics used to study infectious diseases include site-directed mutagenesis, knock-out animals, knock-in animals, conditional knock-outs, inducible knock-out animals, and the use of CRISPR/Cas9 technologies in genomic editing (reviewed in Appleby and Ramsdell 2003; Cui et al. 2018). Forward genetic approaches, in contrast, are phenotype-driven. Upon observation of a phenotype of interest, experiments are conducted to find the underlying genetic cause(s). Several forward genetic approaches have capitalized on varying susceptibility between hosts, as is done with mouse models of infection. Recombinant congenic mouse strains were created by crossing classical inbred mouse strains of differing phenotypes and have been used in several infectious models with complex inheritance (Min-Oo et al. 2003; Roy et al. 2006). Alternatively, chemical mutagenesis using agents such as N-ethylnitrosourea (ENU) has been used by several research groups to introduce genome-wide novel mutations at random. A number of ENU screens revealed the involvement of various genes of immunological importance (reviewed in Caignard et al. 2014; Flaswinkel et al. 2000; Nelms and Goodnow 2001). The genes contributing to the distinct phenotypes can then be mapped and identified, a process that has been expedited with the use of exome and whole genome sequencing (Geister et al. 2018). In this review, we provide a brief overview of the diseases caused by Salmonella, Escherichia, Shigella, and Yersinia. We highlight the contributions of various animal models and genetic approaches to our current understanding of bacterial pathogenesis and of host genetic determinants involved in susceptibility or resistance to infection. Lastly, we briefly discuss the relevance of animal model findings as they relate to human disease.

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Salmonella Overview of the disease Salmonella enterica are one of the leading causes of foodborne infections and remain a major threat for human populations throughout the world. Salmonella enterica are intracellular bacteria that are found in the gastrointestinal tract of mammalian, avian, and reptilian hosts. The genus Salmonella is extremely diverse and comprises at least 2579 serovars within two species (Salmonella bongori and Salmonella enterica). Salmonella enterica infections in humans are responsible for three major clinical syndromes, typhoid fever caused by host-specific Salmonella, a diarrheal disease known as salmonellosis caused by several non-typhoidal Salmonella (NTS) and invasive NTS (iNTS) infection with bacteremia in immunocompromised patients. Typhoid, or enteric fever, is a systemic disease that continues to be a major cause of morbidity and mortality in areas of the world with poor sanitation and hygiene. Approximately 22 million cases are diagnosed per year resulting in more than 433,000 deaths worldwide (Buckle et al. 2012; Crump et al. 2004). Ingestion of contaminated water is the primary cause of infection. As such, typhoid fever is rare in developed countries with approximately 400 cases diagnosed per year and nearly all associated with international traveling (Jong 2012). Typhoid fever is caused by a select group of typhoidal Salmonella serovars restricted to human and higher primate hosts. These include Salmonella enterica serovar Typhi, and serovar Paratyphi A, B, or C. Symptoms include fever, chills, abdominal pain, loss of appetite, and general malaise manifesting 1–3 weeks post-infection. When treated with antibiotics, the infection is typically cleared within a few days. When left untreated, however, symptoms can last up to 4 weeks with a case fatality rate between 12 and 30% (Maskell 2006). A small proportion (1–5%) of those infected individuals become chronic asymptomatic carriers (Levine et al. 1982). Salmonellosis is one of the most common and widely distributed food-borne diseases in Europe and North America (Callejón et  al. 2015). Each year, more than 93.8 million people become sick from consuming food (poultry, eggs, milk, fresh produce) that is contaminated with Salmonella (Majowicz et al. 2010). Salmonellosis in immunocompetent individuals is characterized by a self-limiting gastroenteritis with symptoms of diarrhea, abdominal pain, headache, nausea, and vomiting occurring between 6- and 72-h post-ingestion of contaminated food. These symptoms generally subside within 4–7 days without treatment. The Salmonella serovars most commonly associated with this diarrhoeal disease are Salmonella

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enterica serovar Typhimurium and serovar Enteritidis (Keestra-Gounder et  al. 2015). In contrast, iNTS have emerged as an important cause of bacteremia in immunocompromised individuals (Dhanoa and Fatt 2009). The clinical presentation of iNTS is characterized by a nonspecific febrile illness often without enterocolitis (Gordon et al. 2002; Tennant et al. 2010). The most common cause of iNTS disease worldwide is advanced HIV disease. In sub-Saharan Africa, NTS cause an invasive and severe disease with a case fatality rate estimated at 20% in HIV-infected adults (Feasey et al. 2012). Young children with severe malaria and/or malnutrition are also at increased risk of iNTS (Brent et al. 2006). In high-income countries, NTS bacteremia is rare and occurs in immunocompromised individuals presenting with HIV infection, autoimmune diseases, chronic granulomatous disease, or with the syndrome of Mendelian susceptibility to mycobacterial disease (MSMD) (Gordon 2008; de Beaucoudrey et al. 2010). Pathogenesis The infective doses vary according to the serovars and could be low especially in elderly or immunocompromised individuals (Blaser and Newman 1982). Salmonella have to resist the acidic pH of the stomach before eliciting disease. In mouse models of oral Salmonella Typhimurium infection, 95–99% of the inoculum is eliminated in the stomach (Giannella et al. 1972). Both typhoidal and NTS Salmonella invade the intestinal mucosa preferentially through the microfold cells (M-cells) overlying the Peyer’s patch in the distal ileum (Clark et al. 1994; Jones et al. 1994). Differently from NTS infection, typhoidal serovars do not induce a clinically detectable inflammatory response of the intestine during the initial phase of infection and they present limited intestinal luminal replication. This contrasts with the massive infiltration of neutrophils and fluid into the intestinal lumen resulting in inflammatory diarrhea caused by NTS serovars (Dougan and Baker 2014; Keestra-Gounder et al. 2015). After crossing the epithelium, Salmonella migrate to the lamina propria where they can access the gut-associated lymphoid tissue (GALT) and are taken up by innate immune cells including macrophages, neutrophils, and dendritic cells (Fig. 1a) (Rescigno et al. 2001; Vazquez-Torres et al. 1999). NTS do not usually pass beyond the local lymph nodes in significant number to cause systemic disease in immunocompetent individuals. Following uptake by phagocytes, NTS induce caspase-1-mediated cell death. This initiates the production of IL-1 and IL-18, which subsequently triggers a robust inflammatory response characterized by recruitment of a large number of neutrophils, increased vascular permeability, mucosal edema, necrosis of the ileal mucosa,

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fluid loss, and diarrhea. This contrasts with infection with typhoidal Salmonella where intestinal inflammation is minimal and the bacteria disseminate at systemic sites, either within phagocytic immune cells or directly in the blood, to the mesenteric lymph node, spleen, liver, bone marrow, and gallbladder. Multicellular lesions develop at foci of infection through the recruitment of polymorphonuclear (PMN) leukocytes and bone marrow-derived mononuclear cells (Mastroeni et al. 1992). Typhoidal Salmonella replicate and spread to new foci before re-entering circulation and moving back to the intestinal lumen via secretion in the bile, promoting bacterial shedding. Animal models Mouse models of Salmonella infection have been extensively used to model distinct aspects of the human Salmonella infection in vivo. Typhoidal Salmonella, such as S. Typhi, are highly adapted to humans and do not cause disease in mice. However, Salmonella Typhi infection could be established in humanized immunodeficient mice engrafted with human hematopoietic cells. During systemic infection in non-obese diabetic-scid IL2rγnull and ­Rag2−/−γc−/−, S. Typhi lead to the development of clinical pathology resembling human disease including bacterial replication in the reticuloendothelial system, damage to the splenic and lymphatic architecture, as well as infiltration of immune cells to these organs (Libby et al. 2010; Mian et al. 2011). The most widely used mouse model is one of systemic disease caused by Salmonella Typhimurium which resembles the clinical syndrome of human iNTS with bacteremia. The pathogenesis in mice can be divided into four phases (Mastroeni 2002). During the first phase, Salmonella crosses the epithelium and disseminates to the mesenteric lymph node, spleen, liver, and bone marrow. This phase is marked by a balance between bacterial growth and bacterial clearing by activated phagocytes, primarily neutrophils and macrophages. In the second phase, bacteria undergo exponential growth within immune cells while bacteria killing becomes negligible. The interaction between bacterial virulence and host defense during this phase is crucial for determining the disease outcome in term of mortality. In resistant mice, Salmonella growth is slower and allows for the gradual onset of host defenses as opposed to susceptible mice which are unable to gain control over Salmonella replication. The third phase of pathogenesis involves an innate immune-mediated plateau in bacterial growth. Recognition of Pathogen-Associated Molecular Patterns (PAMPs) by Toll-like receptors (TLRs) and Nod-like receptors (NLRs) triggers a proinflammatory response including the production of antimicrobial peptides, proinflammatory cytokines, reactive oxygen species (ROS), and reactive nitrogen species

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Fig. 1  Overview of disease pathogenesis caused by Salmonella, Citrobacter rodentium, and Shigella spp. a Salmonella preferentially crosses the intestinal epithelium via microfold cells (M-cells). Once across, Salmonella are taken up by phagocytes including macrophages, neutrophils, and dendritic cells. This is followed by recruitment of T and B cells. Non-typhoidal Salmonella subsequently induce caspase-1-mediated cell death, initiating IL-1 and IL-18 production which then triggers a robust inflammatory response. NTS do not usually pass beyond the local lymph nodes in significant number. During systemic typhoid disease in humans or Salmonella Typhimurium infection in mice, Salmonella disseminate to the mesenteric lymph node (MLN) through the lymphatics and blood stream, and then to systemic sites including the spleen and liver. b Oral infection with Citrobacter rodentium causes characteristic A/E lesions in the intestinal epithelium where bacteria tightly attach to host enterocytes and destroy the brush border microvilli. Infection triggers a robust innate and adaptive inflammatory response involving neutrophils, macrophages, dendritic cells, and IL-22-producing group 3 innate lymphoid cells (ILC3). Surface lymphotoxin (LT) on ILC3s signals via the lymphotoxin beta receptor (LTβR) on dendritic cells and epithelial cells to induce IL-23 production. This promotes ILC3s to produce IL-22, which signal via the IL-22R on epithelial cells and drives the expression of antimicrobial peptides such as RegIIIβ

and RegIIIγ through a STAT3-dependent mechanism. In addition, IgG antibodies from B cells, IL-17-producing Th17 cells, and IL22secreting Th22 cells play a central role in the clearance of Citrobacter rodentium infection and in driving host resistance. Disease severity can range from self-limiting colitis to lethal diarrhea depending on the genetic background of the host. Rspo2 is robustly induced by the stroma specifically in infected susceptible mouse strains, resulting in pathological activation of WNT signaling, generation of a poorly differentiated colonic epithelium, and intestinal dysfunction. c Two alternative pathways have been described for the invasion of colonic mucosae by Shigella spp. The first pathway necessitates entry through M-cells. The second pathway proceeds through the direct invasion of epithelial cells. After a brief residence into the entry vacuole, Shigella ruptures the vacuole, accessing the cytosol of epithelial cells. Using actin comets, Shigella moves in the cytosol and induces the formation of protrusions. Bacteria found in protrusions are captured into double membrane dissemination vacuoles. The rupture of dissemination vacuole resembles that of entry vacuole although it also displays unique features. Invasion of epithelial cells releases large amounts of IL-8. This leads to the recruitment of neutrophils to the infection foci. Both mucosal invasion pathways lead to the penetration of Shigella within the submucosae. Infected macrophages are killed through pyroptosis, triggered by high amount of IL-1β and IL-18

(RNS), as well as the regulation of intracellular iron levels and coordination of various processes of cell death. The fourth and final phase features an adaptive immunemediated increase in bacterial clearing. B-lymphocytes generate antibodies against lipopolysaccharide (LPS), flagellin, and other outer membrane proteins. B cells are also important for initiating a T-lymphocyte immunity through antigen presenting functions (Nanton et al. 2012; Ugrinovic et al. 2003). The expansion and differentiation of T-lymphocytes is critical during Salmonella clearance. Mice with deficient T-cell immunity, such as mice lacking mature CD4+ TCR-alpha beta cells, TCR-alpha beta cells, or deficient in CD28, are highly susceptible to infection with Salmonella of attenuated virulence (Hess et al. 1996; Mittrucker et al. 1999). As with other intracellular

bacterial and viral infections, a Th1 bias is observed during infection with Salmonella. This promotes the production of proinflammatory cytokine IFN-γ necessary for Salmonella clearance (Pie et al. 1997). Throughout the course of infection, host resistance depends on the genetic background of the mice. Classical inbred strains of mice can be classified into three distinct categories with respect to their susceptibility to Salmonella Typhimurium infection. 129 substrains (129S1, 129S6, 129X1) are extremely resistant to infection compared to A/J or CBA/J mice that present an intermediate susceptibility phenotype. Other strains such as C57BL/6J, BALB/ cJ, FVB/J, and C3H/HeJ are extremely susceptible to infection and all succumb within the first week of infection (Roy and Malo 2002). Mouse susceptibility to infection with

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Salmonella Typhimurium is determined by various mutations and will be discussed later. Although widely accepted and useful as model for studying iNTS or typhoid fever, Salmonella Typhimurium in mice does not naturally cause intestinal inflammation and enterocolitis. Experimental oral infection or inoculation of ileal ligated loops in calves are recognized models of Salmonella-induced enteric disease that parallels clinical disease and pathological changes observed in human salmonellosis caused by NTS (Costa et al. 2012) These models have been used to study pathologic changes occurring during infection and for the characterization of Salmonella virulence factors. However, working with a large animal model presents several limitations (housing, cost, high interindividual variability). A mouse model for Salmonella Typhimuriuminduced enterocolitis has been developed by the group of WD Hardt that uses pretreatment of mice with a single dose of streptomycin to diminish the colonization resistance barrier (reviewed in Wotzka et al. 2017). The antibiotic disrupts the normal intestinal flora and Salmonella Typhimurium efficiently colonizes the large intestine and triggers a severe acute diffuse inflammation of the cecum (Barthel et al. 2003; Stecher et al. 2005). Chronic models of infection have been also developed to study Salmonella pathophysiology later during the course of infection. In these models, the mice do not succumb to the infection and carry the bacteria for a prolonged period of time in the reticuloendothelial system and mesenteric lymph nodes. The chronic models use Salmonella Typhimurium of attenuated virulence in susceptible mice, sublethal infection with Salmonella Typhimurium in resistant mice or sublethal infection with Salmonella Enteritidis (Caron et al. 2006; Monack et al. 2004). The similarities in pathophysiology between mice and humans during chronic infection make mice a valuable model for future studies of the factors leading to chronic carriage, a phenomenon that remains poorly understood. Thus, despite their limitations with modeling human diseases, Salmonella mouse models have been invaluable to understand the pathogenesis of Salmonella infection in vivo. Genes/loci identified in animal models Both reverse and forward genetics have been used successfully to identify loci/genes that play an important role in vivo in the host response to systemic Salmonella infection in mice. Genetic studies have helped gain insight into the function of individual proteins along with overall immunopathology during infection and showed the central role of innate and adaptive immunity in determining the outcome of infection. The list of genes resulting in Salmonella systemic disease in mice and humans is growing and has been reviewed previously (Gilchrist et al. 2015a; Vidal et al.

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2008). We will discuss here selected examples of major Salmonella susceptibility genes identified by forward genetics in classical inbred mice, recombinant congenic strains, and in chemical-induced mouse mutants. In the 1980s and 1990s, the natural occurring range in innate susceptibility to Salmonella infection in classical inbred mice provided a means to investigate underlying genetic determinants of susceptibility. This was the case for two mouse strains carrying either the Ity (C57BL/6J) or Lps (C3H/HeJ) locus. C57BL/6J and C3H/HeJ are extremely susceptible to infection with Salmonella Typhimurium as measured by shorter survival time following infection and high bacterial load in spleen and liver. Positional cloning using multiple crosses between resistant and susceptible mouse strains and different mapping and cloning approaches has led to the discovery of solute carrier family 11 member 1 (Slc11a1 initially known as Nramp1) for Ity, one of the earliest genes to be isolated by positional cloning. Slc11a1 has pleiotropic effects and confers resistance to other intracellular pathogens including Mycobacterium bovis and Leishmania donovani. Slc11a1 is recruited to the phagosomal membrane during infection contributing to Salmonella containing vacuole (SCV) maturation and mediating iron depletion from the phagosome (reviewed in Vidal et al. 2008). Using a similar forward genetics approach, Toll-like receptor 4 (Tlr4) was identified as the gene underlying the Lps locus in C3H/HeJ mice (Poltorak et al. 1998; Qureshi et al. 1999). TLR4 interacts with lipopolysaccharide (LPS), a structural component of the outer membrane of gram-negative bacteria. The absence of TLR4 decreased the bacterial killing ability of macrophages, resulting in increased bacterial burden throughout the reticuloendothelial system, and, ultimately, decreased survival (Weiss et al. 2004). The discovery of human toll-like receptor (Medzhitov et al. 1997) and the identification of the molecular basis of the Lps mutation represented a significant advance in defining the fundamental mechanisms of cellular activation by LPS and in establishing the importance of the TLR family as a pathogen recognition system for all major classes of microbes (O’Neill et al. 2013). Red blood cell (RBC) defects in mice caused by mutation in the genes Pklr (pyruvate kinase liver and red blood cell) identified in the recombinant congenic mice AcB61 and Ank1 (ankyrin 1) identified in different screens with the mutagen N-ethyl-N-nitrosourea (ENU) were shown to confer both resistance to malaria (Greth et al. 2012; Min-Oo et al. 2003) and susceptibility to Salmonella infection (Roy et al. 2007; Yuki et al. 2013). The reasons underlying the increased susceptibility to infection with RBC defects are not completely understood, but may be related to the severity of the RBC hemolysis resulting in anemia, heme-mediated phagocyte dysfunction (impaired oxidative burst), and/or iron overload (Cunnington et al. 2011; Roy et al. 2007).

Enterobacteria and host resistance to infection

More recently, ENU chemical mutagenesis was used to overcome the limited genetic diversity present in inbred mouse strains. This strategy was successful to identify novel Salmonella resistance genes which uncover additional pathways and mechanisms (Caignard et al. 2014). This is the case for Usp18 (ubiquitin specific peptidase 18) which negatively regulates type I interferon signaling and has deISGylation activity for ISG15 (interferon stimulated gene 15) (Richer et al. 2010). Mutation within USP18 in mice also confers susceptibility to infection with Mycobacterium tuberculosis (Dauphinee et al. 2014). ENU mutation within the transcription factor Stat4 (signal transducer and activator of transcription 4) emphasizes the importance of type II IFN pathway to fight Salmonella infection (Eva et al. 2014). STAT4 is known to mediate IFN-γ release from natural killer and T cells in response to IL-12. ENU mutagenesis has also identified an uncharacterized gene (Fam49b) for controlling susceptibility to Salmonella infection. FAM49B shares a conserved domain for RAC1 (Rac Family Small GTPase 1) binding present in CYFIP (Cytoplasmic FMR1 Interacting Protein 1) proteins. CYFIP proteins are part of the WAVE regulatory complex (WRC) that regulates actin filament remodeling via the activation of RAC1, a process essential for cellular engulfment of Salmonella into SCVs. FAM49B binds to RAC1 and negatively regulates RAC1driven actin remodeling, attenuating bacterial entry into epithelial cells, phagocytosis as well as phagocyte-mediated Salmonella dissemination. FAM49B was renamed CYRI1 (CYFIP-related RAC1 Interacting protein 1) to denote its function as a negative regulator of RAC1-driven cellular effects (Yuki et al. 2017). Relevance to human disease The suitability of mouse models to the study of systemic Salmonella infection in humans can be garnered from similarities in pathology across organisms but also from shared genes and pathways involved in immunity. In some instances, studies in mice have provided candidate genes to explore genetic variation in specific human cohorts for susceptibility to iNTS and other intracellular pathogens, in other cases mice were used to validate and study the function of genes identified in human population studies. The contribution of genetic variation in increasing risk of developing typhoid fever was demonstrated using candidate gene association studies in humans and identified genes within the major histocompatibility complex (HLA-DRB1 and TNF), TLR4, and PRKN (Parkin RBR E3 Ubiquitin Protein Ligase), significantly associated with typhoid fever (Ali et al. 2006; Bhuvanendran et al. 2011; Dharmana et al. 2002; Dunstan et al. 2007, 2014). All these genes/loci were initially shown to have an impact on resistance to systemic infection in mice with the exception of PRKN.

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The host genetic background is also playing an important role in the vulnerability of an individual to iNTS (Gilchrist et al. 2015a). Association between RBC defects, malaria, and iNTS has been reported in humans. This is well illustrated in human hemoglobinopathies and red blood cell enzymatic defects (beta-Thalassemia and Sickle cell disease) known to confer protection to malaria in human populations, while at the same time conferring susceptibility to iNTS (Takem et al. 2014). Immunodeficiency syndromes in humans have provided further evidence of the conservation of immunopathology between mice and humans. Defects in the IL-12/STAT4/IFNγ axis associated with MSMD cause an increased risk of disseminated NTS (van de Vosse et al. 2009). STAT4 was also identified using a genome-wide association study as a susceptibility locus for iNTS disease in African children (Gilchrist et al. 2015b). Other immune deficiencies including chronic granulomatous disease (defect in NADPH oxidase activity involving the genes CYBB and NCF2), X-linked hyper-IgM syndrome (defect in CD40LG/ CD40 signaling), X-linked agammaglobulinaemia (defect in the gene BTK) have also shown increased iNTS susceptibility in both mice and humans demonstrating the value of mice when modeling various stages of infection (Gilchrist et al. 2015a; Vidal et al. 2008).

Escherichia Overview of the disease Although commensal E. coli strains are natural inhabitants of the human gastrointestinal tract, several pathogenic variants of E. coli exist, many of which can cause significant health complications in humans and animals due to their ability to acquire virulence factors and transmit via the fecal–oral route through contaminated food and water (reviewed in Kaper et al. 2004). A recent report by the World Health Organization estimated that diarrheal diseases, in which pathogenic E. coli is a major contributor, are responsible for 550 million illnesses and 230,000 deaths every year (Havelaar et al. 2015). Diarrheagenic E. coli are typically divided into six major pathotypes (reviewed in Croxen et al. 2013 and; Kaper et al. 2004): enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enteroaggregative E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and diffusely adherent E. coli. In this review, we focus on the human pathogens EPEC and EHEC, which cause significant morbidity and mortality worldwide. EPEC is a leading cause of infantile diarrhea particularly in developing countries while EHEC causes bloody diarrhea mainly in children and the elderly in developed countries (Nataro and Kaper 1998). EHEC is distinguished from EPEC by the expression of the highly potent Shiga toxin (Stx), which is associated with the development of hemorrhagic colitis and/or hemolytic uremic

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syndrome (Nataro and Kaper 1998). EHEC and EPEC are also distinguished by their tissue tropism: EHEC colonizes the large intestine while EPEC colonizes the small intestine. Pathogenesis EPEC and EHEC are extracellular mucosal pathogens that share a unique mechanism of colonization characterized by the formation of attaching and effacing (A/E) lesions in the intestinal epithelium where bacteria intimately attach to the plasma membrane of host enterocytes, destroy the brush border microvilli, and induce cytoskeletal rearrangements underneath the adherent bacteria (reviewed in Kaper et al. 2004). The development of A/E lesions relies on the highly conserved locus of enterocyte effacement (LEE) pathogenicity island, which encodes the type three secretion system (T3SS) and effector proteins required for pathogenesis (reviewed in Collins et al. 2014). These effector proteins perform critical roles when translocated into host cells by subverting host defenses and cellular processes which enable the bacteria to colonize, multiply, and cause disease (reviewed in Wong et al. 2011). The genes encoding effector proteins vary considerably among A/E pathogens (Muller et al. 2009), and it is this variation that likely reflects their difference in virulence phenotypes. EPEC and EHEC outbreaks occur worldwide with different clinical outcomes ranging from asymptomatic to severe or lethal disease. Experimental infection of adult human volunteers with a wild-type strain of EPEC caused substantial differences in the response to infection within the volunteer cohort (Tacket et al. 2000). Similarly, only a fraction of patients infected with EHEC develop hemolytic uremic syndrome in which Stx travels to the kidney through the bloodstream and inhibits protein synthesis in endothelial cells, inducing renal inflammation (Karch 2001). The damage this causes is characterized by hemolytic anemia, thrombocytopenia, and acute kidney failure. Despite the data suggesting that host genetics can influence disease outcome to infection by A/E pathogens, there are few human studies identifying host genetic loci involved in resistance or susceptibility to diarrheagenic E. coli. One study demonstrated that individuals with the AA genotype at the -251 position of the IL-8 gene promoter were associated with increased risk of diarrhea due to enteroaggregative E. coli and greater fecal IL-8 production (Jiang et al. 2003). Another demonstrated that individuals who are genetically predisposed to produce high levels of IL-10 due to a major single nucleotide polymorphism (SNP) in the IL-10 promoter are likely to develop diarrhea when exposed to enterotoxigenic E. coli (Flores et al. 2008). Other SNPs such as those within the lactoferrin and osteoprotegerin genes were shown to be associated with increased susceptibility to enteric pathogens including EPEC and EHEC although these findings did not correlate disease

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to any specific pathogen (Flores and Okhuysen 2009). The precise mechanism of diarrhea is not fully understood but it likely involves multiple mechanisms including impaired ion secretion and transport due to loss of absorptive surfaces resulting from microvillus effacement, increased intestinal permeability due to disruption of tight junctions, and inflammation (reviewed in Croxen et al. 2013; Kaper et al. 2004). Animal models Although no single animal model perfectly mimics the natural disease process caused by A/E pathogens in humans, several animal models have been developed to study A/E pathogenesis. The nematode Caenorhabditis elegans is a simple in vivo model that has been shown to be susceptible to infection by EPEC and EHEC (Anyanful et al. 2005; Chou et al. 2013; Mellies et al. 2006). This has allowed for the identification of novel genes and small signaling molecules secreted by E. coli that together regulate production of virulence factors and toxins. In addition, several recent studies used the C. elegans model to investigate the antibiofilm effects of various plant alkaloids and essential oils against EHEC as alternatives to antibiotic strategies (Kim et al. 2016; Lee et al. 2017), since certain antibiotics may worsen disease symptoms or induce Stx production (Serna and Boedeker 2008). EHEC and rabbit EPEC (REPEC) infection of rabbits cause important human-related features of disease such as severe diarrhea and intestinal inflammation (Ritchie and Waldor 2005; Robins-Browne et al. 1994). Early studies in rabbits were used to explore the effects of virulence factors including Stx in the development of diarrhea. Furthermore, rabbit models led to the discovery of factors essential for mediating intestinal colonization and survival (Ritchie and Waldor 2005; Robins-Browne et al. 1994). REPEC displays similar pathological characteristics and tissue tropism with human EPEC (Robins-Browne et al. 1994), and shares a high degree of homology to EPEC LEE (Tauschek et al. 2002). This has facilitated our understanding of the influence of many LEE genes and effector proteins such as EspA and EspB on EPEC virulence and A/E lesion formation (Abe et al. 1998; Marches et al. 2000). Gnotobiotic piglets and cattle are additional animal models that are used sporadically to study virulence factors and intestinal manifestations of A/E disease. Readers are directed to Law et al. 2013 and Ritchie 2014 for comprehensive reviews on the various in vivo models to study EPEC and EHEC infections. Limitations to these models exist, in that powerful genetic tools are lacking. Mice, however, provide an excellent system for genetic research due to the ease of manipulating the mouse genome to model human disease. Mice are naturally resistant to infection with EPEC and EHEC, and often do not develop signs of intestinal disease (Mundy et al. 2005). Citrobacter rodentium is a mouse-specific pathogen that shares

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several key pathogenic mechanisms with EPEC and EHEC, and expresses many of the same LEE-related genes and effector proteins required to form A/E lesions (Collins et al. 2014). As a result, C. rodentium has been used extensively to elucidate the virulence roles of LEE-encoded and T3SStranslocated effector proteins as well as non-LEE-encoded proteins. Moreover, a Stx-expressing C. rodentium strain was recently generated to serve as a more relevant model of EHEC infection (Mallick et al. 2012). These studies significantly advanced our understanding of the molecular basis of A/E disease including how effector proteins interfere with host cell processes. Hence, C. rodentium is widely considered as an outstanding small-animal model to study A/E virulence and mechanisms of disease in vivo. Oral infection with C. rodentium causes colitis and characteristic thickening of the mucosa and elongation of the colonic crypts called transmissible murine crypt hyperplasia (Luperchio and Schauer 2001; Mundy et al. 2005). Importantly, disease severity can range from self-limiting colitis to lethal diarrhea and inflammation depending on the genetic background of the host (Borenshtein et al. 2007; Mundy et al. 2005). C. rodentium initially colonizes the cecum within the first day before progressing to the distal colon 2–3 days post-infection. This is followed by a marked increase in C. rodentium growth in the colon, resulting in dysbiosis and major alterations to the overall composition and diversity of the commensal microbiota. The infection clears as adherent bacteria and colonized epithelial cells get shed into the lumen until complete clearance in the stool occurs 3–4 weeks post-infection (Collins et al. 2014; Mundy et al. 2005; Wiles et al. 2004). Both innate and adaptive immune responses are essential for protection against C. rodentium; infection triggers robust inflammatory responses in the colon involving infiltration of immune cells such as neutrophils, macrophages, and dendritic cells and the induction of IL-17-producing Th17 cells and IL-22-producing group 3 innate lymphoid cells (ILC3) (Fig. 1b) (Collins et al. 2014; Geddes et al. 2011). IL-22, in particular, has been shown to be critical in the early response to C. rodentium by driving the expression of antimicrobial peptides in the colonic epithelium and promoting epithelial barrier integrity (Zheng et al. 2008). Complete resolution of infection is dependent on functional B cells and C ­ D4+ T cells (Simmons et al. 2003; Vallance et al. 2002). Notably, IgG antibodies, IL-17-producing Th17 cells, and IL-22-secreting Th22 cells play a central role in the clearance of C. rodentium infection and in driving host resistance (see Collins et al. 2014 and; Koroleva et al. 2015 for reviews on the contributions of key immune cells, cytokines, and signaling pathways to the immune response to C. rodentium). Recent studies have investigated the role of the intestinal microflora in mediating gut homeostasis and virulence of C. rodentium infection. Indeed, transfer of the microbiota from

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resistant to susceptible mice prevented C. rodentium-induced mortality (Ghosh et al. 2011) while depletion of kanamycinsensitive commensals during the peak of infection, but not of metronidazole or vancomycin, was shown to displace C. rodentium from the colonic mucosa, indicating that enteric pathogens may rely on specific commensals for colonization of mucosal surfaces (Mullineaux-Sanders et al. 2017). Further work is necessary to understand the mechanisms behind host–pathogen–microbiota interactions to improve therapeutic approaches to infection with A/E pathogens. Genes/loci identified in animal models The C. rodentium mouse model has led to the discovery of pivotal functions of immune cell subsets such as ILC3s and Th17 cells, and is often used to investigate the host immune response to pathogenic E. coli infections. Studies in mice with targeted deletions of certain components of the immune system have shed light on the different compartments of the immune system and signaling pathways that are important for disease pathogenesis (reviewed in Koroleva et al. 2015). For example, studies using knock-out mice or biochemical inhibitors have revealed that mice deficient in IL-23, IL-22, LTβR, MyD88, STAT3, ILC3s, and mature T and B cells are highly susceptible to C. rodentium infection (Koroleva et al. 2015). Furthermore, a study using mouse strains from the advanced inbred BXD panel and infection with Stx-producing E. coli was conducted to identify host genetic factors that may be determinants for EHEC-related disease outcome (Russo et al. 2015). The authors observed significant differences in colonization levels between mouse strains, and identified several candidate genes including Acad8, Bmper, Pde4a, Panx1, and Dnmt1 within a quantitative trait locus that is highly associated with variation in colonization. Our group and others have identified a number of inbred mouse strains that are hyper-susceptible to C. rodentium infection compared to resistant C57BL/6 mice: C3H/HeJ, C3H/HeOuJ, FVB, and AKR/J (Borenshtein et al. 2007; Papapietro et al. 2013; Vallance et al. 2003). Linkage analysis using F2 crosses between resistant and susceptible mice yielded two major genetic loci entitled Cri1 and Cri2, in which the former effectively controls mortality during C. rodentium infection (Diez et al. 2011; Papapietro et al. 2013; Teatero et al. 2011). We subsequently used a forward genetics approach to identify the R-spondin 2 (Rspo2) gene as a major determinant of susceptibility to C. rodentium infection underlying the Cri1 locus (Papapietro et  al. 2013). Rspo2 is a potent enhancer of the canonical Wnt signaling pathway, which plays a crucial role in regulating intestinal epithelial cell fate, determination, and proliferation (Jin and Yoon 2012; van der Flier and Clevers 2009). Rspo2 is robustly induced specifically in infected susceptible mouse strains, leading to pathological activation of Wnt signaling,

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generation of a poorly differentiated colonic epithelium, and impaired intestinal ion transport activities (Kang et al. 2018; Papapietro et al. 2013). These studies uncovered a novel pathway involved in the development of diarrhea and intestinal dysfunction through Rspo2-mediated disruption of intestinal differentiation. Relevance to human disease Notably, gene fusions that bring RSPO2 and another member of the R-spondin family, RSPO3 under the control of strong heterologous promoters potentiates Wnt signaling and is associated with colorectal cancer development in humans (Seshagiri et al. 2012). In addition, a recent study found that increased Rspo3 expression upon infection with the enteric pathogen Helicobacter pylori may increase the risk of gastric cancer by causing hyperproliferation and gland hyperplasia (Sigal et al. 2017). This, together with our own work with Rspo2, suggests that R-spondin-mediated signaling has broad relevance in inflammation-associated intestinal disease. While published genetic studies in mice are few, genome-wide approaches with new and evolving technologies may show promise in uncovering novel genes or loci involved in diarrheal disease susceptibility.

Shigella Overview of the disease Shigella spp. naturally infect only humans. The four major serogroups are called Shigella dysenteriae, flexneri, boydii, and sonnei and are considered pathovars of Escherichia coli. Shigella infection, also known as shigellosis, causes an acute large intestine infection with a prominent inflammatory component. The symptoms range from mild watery diarrhea to severe dysentery. The latter condition is characterized by abdominal cramps, fever, and liquid stools containing blood and mucus. Shigella spp. are mostly transmitted from person-to-person through the fecal–oral route but food and water contamination are also encountered (reviewed in Phalipon and Sansonetti 2007; Schroeder and Hilbi 2008). According to data from the Centers for Disease Control and Prevention, Shigella spp. are estimated to cause 80–165 million illnesses resulting in 0.6 million deaths per year (Bowen 2017). Most cases are due to S. flexneri and occur in young children from developing countries. By contrast, S. sonnei is more frequently encountered in developed countries (Thompson et al. 2015). Due to the emergence of antibioticresistant strains and the prevalence of the disease, shigellosis is a threat to public health (Sansonetti 2006). This is illustrated by the fact that the World Health Organization has included Shigella spp. on the list of “bacteria for which novel

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antibiotics are urgently needed” (World Health Organization 2017). Pathogenesis At ambient temperature, Shigella is non-invasive (Falconi et al. 1998; Maurelli and Sansonetti 1988). During the transit through the gastrointestinal tract, Shigella invasiveness increases due to expression of virulence genes at 37 °C. The infectious dose (ID) of Shigella spp. ranges between 10 and 500 bacilli in healthy human volunteers (reviewed in Kothary and Babu 2001). However, the percentage of volunteers with symptoms for comparable ID varied widely across studies, indicating that these numbers must be interpreted with care. Notwithstanding that, the IDs reported for other enteropathogens, such as Vibrio cholerae, Salmonella Typhimurium, and EPEC, are systematically higher than for Shigella spp. (Kothary and Babu 2001). This has been proposed to stem from the resistance of Shigella spp. to acidic pH (Gorden and Small 1993). The invasiveness of Shigella spp. was suggested to be primed by exposure to bile salt (Barta et al. 2012; Pope et al. 1995) and may at least partly explain the tropism of Shigella spp. for the large intestine. The proposed model describing the invasion of this organ by Shigella spp. relies on data compiled from in vitro and in vivo studies performed in both the small or the large intestine of different animals (reviewed in Cossart and Sansonetti 2004; Schroeder and Hilbi 2008). In this model, Shigella spp. invade the large intestine mucosae using microfold cells (M-cell) as Trojan horses. Bacteria then would use a process akin to transcytosis to access the underlying lymphoid tissue. Their survival in this hostile environment might be facilitated by their capacity to kill macrophages through pyroptosis. Some bacteria eventually escape the lymphoid tissue by invading colonic epithelial cells (colonocytes) on their basolateral side (Cossart and Sansonetti 2004; Schroeder and Hilbi 2008). Recently, the apical invasion of colonocytes in the vicinity of crypts openings has been observed in the Guinea pig large intestine (Arena et al. 2015). These results pose the question of the coexistence of an apical and basolateral entry route. Finally, another hallmark of shigellosis is the massive IL-8-induced recruitment of neutrophils that are essential to block the penetration of S. flexneri within the deeper layer of the tissue (Perdomo et al. 1994; Sansonetti et al. 1999). This phenomenon might explain the observation that S. flexneri is overall kept away from the bottom of colonic crypts in the Guinea pig as well (Arena et al. 2015). The infectious life cycle of Shigella spp. in colonocytes can be broken down in three stages (reviewed in CampbellValois and Pontier 2016): (1) entry through formation of a phagosome-like vacuole, which is ultimately ruptured; (2) residence in the cytoplasm where most replication events and actin comet-based motility take place; (3) cell-to-cell

Enterobacteria and host resistance to infection

spread, which enables the transfer of a bacterium from an initially infected cell to a neighboring cell (Fig. 1c). Bacteria can iteratively repeat stages two and three to progressively invade an increasing number of colonocytes. The central element of the virulence of Shigella spp. is the T3SS. Its most important component is the Type Three Secretion Apparatus (T3SA), also known as the injectisome or syringe, which secretes approximately 30–40 substrates in the S. flexneri strain M90T (Pinaud et al. 2017). Many of these substrates are effectors translocated within host cells to favor the infection. For example, some effectors stimulate Shigella spp. uptake by non-phagocytic cells, while others enable the escape from the vacuole, or block inflammation (Ashida et al. 2015; Campbell-Valois and Pontier 2016; Mattock and Blocker 2017). The T3SA of S. flexneri is active during entry and cell-to-cell spread, while it is inactive in the host cytosol (Campbell-Valois et al. 2014). Most virulence factors including T3SS components and proteins implicated in the cytosol motility are encoded on a large plasmid of approximately 200 kbp known as the invasion or virulence plasmid (Buchrieser et al. 2000; Venkatesan et al. 2001). Most of Shigella spp. pathogenesis was studied with the serogroup S. flexneri. These findings are expected to hold true for other serogroups although differences with S. sonnei have emerged (Anderson et al. 2017; McVicker and Tang 2016). Animal models A wealth of animal models has been used to study the pathogenesis of Shigella spp. but none have been able to reproduce all the hallmarks of the human disease. The virulence of Shigella strains in mammals has been studied in both intestinal and non-intestinal models. For example, the keratoconjunctivitis assay in the Guinea pig (Okamura et al. 1983; Sereny 1957) or the pulmonary challenge assay in the mouse (van de Verg et al. 1995; Voino-Yasenetsky and Voino-Yasenetskaya 1962) have been classically used to evaluate the virulence of strains. More recently, mice were shown to succumb to intravenous and intraperitoneal infections with S. flexneri within 2–7 days (Li et al. 2017; Yang et al. 2014). Interestingly, intraperitoneal infection led to the colonization of the large intestine provoking symptoms similar to shigellosis (Yang et al. 2014). However, bacteria were found mostly around the submucosal muscle layer rather than in the mucosae as observed when infection proceeded through the luminal face (Yang et al. 2014). The most relevant animal models are those where Shigella pathogenesis is studied in the intestine using intragastric infection (IGI) or intrarectal infection (IRI). Primates such as rhesus macaque reproduce well human shigellosis through IGI (Formal et al. 1991; Sansonetti and Arondel 1989), but their usage is limited due to financial and ethical considerations. The common laboratory mouse strains tested (BALB/c in most

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studies; C57BL/6 in Li et al. 2017) are refractory to the colonization of the large intestine by S. flexneri and do not display the inflammatory response that characterizes shigellosis (Cossart and Sansonetti 2004; Singer and Sansonetti 2004). Colonization of the large intestine by S. flexneri has, however, been observed in newborn mice (Fernandez et al. 2003; Martino et al. 2005) or in adult mice pre-treated with streptomycin (Fernandez et al. 2003; Martino et al. 2005) or human IL-8 (Singer and Sansonetti 2004), or harboring human colon xenografts (Sperandio et al. 2008; Zhang et al. 2001). Interestingly, it was also recently shown that S. sonnei is capable of colonizing the large intestine of mouse about three orders of magnitudes more efficiently than S. flexneri by IGI (Anderson et al. 2017). Due to the difficulties of obtaining a relevant mouse model, alternative animal models have been exploited. For example, the rabbit ileal loop has provided invaluable insights on shigellosis (Puhar et al. 2013; Sansonetti et al. 1995; Schnupf and Sansonetti 2012; West et al. 2005). The main limitations of this model are that the ileum is infected instead of the large intestine and that it requires surgical training. IRI of the Guinea pig colon is a model that has recently gained popularity (Arena et al. 2015, 2017; Shim et al. 2007). The characteristics of infected tissue in the Guinea pig resemble those described for human shigellosis (Shim et al. 2007). However, a large inoculum (e.g., 1 × 109–1 × 1010 colony-forming units in Guinea pigs versus 500 in humans) is necessary to obtain bacteria in the mucosae of the majority of tested animals (Arena et al. 2015; Shim et al. 2007). Finally, the Guinea pig is not currently compatible with sophisticated genetic studies. Genes/loci identified in animal models The paucity of genetically tractable animal models allowing studying Shigella spp. infection has considerably limited our knowledge of host resistance mechanisms. Mice with human colon xenograft have, however, provided interesting insights on this matter. Indeed, specific antimicrobial peptides, such as the β-defensins, are upregulated during Shigella infection (Sperandio et al. 2008). Lower expression of β-defensins 1 and 3 correlated with the penetration of bacteria in the deeper layer of the mucosae. A handful of studies using knock-out mice have also been reported. For example, caspase-1 knock-out mice demonstrated the role of IL-1β and IL-18 in Shigella-induced inflammation and its critical role in resolving the infection (Sansonetti et al. 2000). More recently, three groups have independently identified the guanylate binding protein 1 (GBP1) as a key factor restricting Shigella cell-to-cell spread (Li et al. 2017; Piro et al. 2017; Wandel et al. 2017). Furthermore, Feng Shao and coll. have identified a locus encoding a group of several GBPs (GBP1, 2, 3, 5, and 7) as an important host resistance factor to S. flexneri infection. Mice deficient in these GBPs

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died upon intravenous and intraperitoneal infection with the attenuated ΔipaH9.8 strain while wild-type mice did not (Li et al. 2017). The bacterial E3 ubiquitin ligase IpaH9.8 has therefore apparently evolved to degrade the GBPs, therefore counteracting their capacity to restrain the cell-to-cell spread and the growth of intracellular S. flexneri (Li et al. 2017). Relevance to human disease The only known natural host of Shigella spp. is Homo sapiens. This is illustrated by the observation that most animal models must be challenged with a larger inoculum than humans to obtain robust colonization of the large intestine. As of today, there is no ideal model of shigellosis. While the newly developed Guinea pig model certainly offers the highest level of resemblance to human pathogenesis among non-primate models (Arena et al. 2015; Shim et al. 2007), other models, such as the rabbit ileal loop (Puhar et al. 2013; Sansonetti et al. 1995; Schnupf and Sansonetti 2012; West et al. 2005), or more recently the intraperitoneally injected mice (Li et al. 2017), have allowed the community to acquire invaluable insights about Shigella pathogenesis. The emergence of mouse models based on the expression of human IL-8, pretreatment with antibiotics, use of S. sonnei, or a combination of those are very promising (Anderson et al. 2017; Martino et al. 2005; Singer and Sansonetti 2004). They could put the powerful murine genetic toolbox at work for studying the host genetic component of shigellosis.

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rodents and their fleas and can remain unnoticed for decades in the absence of human cases. Human contamination occurs accidentally when fatal infection in rodents forces infected fleas to abandon their dead hosts and seek new ones (Perry and Fetherston 1997). Human plague can take two forms. Bubonic plague results from intense multiplication of bacteria and inflammation in the lymph node draining the bite site which becomes swollen and very painful. In most cases, the bacteria disseminate in the blood stream, induce septicemia, and infect other organs such as the liver and lungs (Du and Wang 2016; Perry and Fetherston 1997). Death occurs in 40–70% of cases in the absence of treatment. Pulmonary plague results from the inhalation of infectious aerosols and is the most rapidly fatal form of the disease with high fever, chest pain, dyspnea and coughing, and death in 40% of cases despite treatment. Because it is highly contagious and virulent, Y. pestis is considered one of the most likely bacteria to be used as a bioterrorism agent (Inglesby et al. 2000). Pathogenesis Early after infection, Y. pestis establishes an immune-suppressive environment, which requires two virulence factors induced at 37 °C in the mammalian environment: the tetra-acylated form of lipid A which is less stimulatory to

Yersinia Overview of the disease The genus Yersinia is composed of 18 species including three (Y. pestis, Y. pseudotuberculosis and Y. enterocolitica) that cause disease in humans. Y. pseudotuberculosis and Y. enterocolitica are food-borne pathogens and cause self-limiting enteric infections known as yersiniosis. Sporadic cases of yersiniosis have been observed worldwide although more frequently in Europe and North America (Dube 2009). In contrast, Yersinia pestis is responsible for plague, the most severe of all human bacterial infections which has caused several devastating pandemics during human history (Bramanti et al. 2016). The second pandemic, referred to as the Black Death, occurred in 1346–1353 and killed a quarter of the Western European population. During the last and still on-going pandemic, the etiological bacterium was isolated in Hong Kong in 1894 by Alexandre Yersin, a disciple of Louis Pasteur. Three years later, Paul-Louis Simond demonstrated that Y. pestis is transmitted between rats and from rats to humans by flea bites (Perry and Fetherston 1997). Y. pestis is primarily an infection of rodents of which over 200 species can be infected in the wild. The infection circulates between

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Fig. 2  Interactions between Yersinia pestis and host immunity. Y. pestis type three secretion system delivers into the cytosol several effector proteins which inhibit bacteria phagocytosis (YpkA), NF-κB and MAPK signaling pathways (YopP, YopJ) and caspase-1 (YopM). YopM stimulates the production of anti-inflammatory IL-10. Conversely, the needle protein YscF activates TLR4 which results in the production of IFN-β and proinflammatory cytokines such as IL-6. Bacteria internalized in Yersinia containing vacuoles are sensed by TLR7 which activates the NF-κB pathway. Also shown are other genes mentioned in the text

Enterobacteria and host resistance to infection

TLR4, and the T3SS which allows extracellular bacteria to deliver immunomodulating factors into phagocytic cells (Fig. 2) (Du and Wang 2016; Li and Yang 2008). These factors, known as Yersinia outer proteins (Yops), disrupt host immune response signaling and induce programmed cell death in macrophages, but not in neutrophils (Spinner et al. 2010). In pneumonic plague, this anti-inflammatory state lasts for 36–48 h and allows bacteria to multiply rapidly in the lung. In a second phase, a strong proinflammatory response is engaged, with activation of proinflammatory cytokines and chemokines (such as IL-6, IL-12p70, TNF-α, KC, and MCP-1), and active and continued recruitment of neutrophils which phagocyte and kill bacteria but do not undergo apoptosis and accumulate in the lung, contributing to massive lung tissue damage (Bubeck et al. 2007; Du and Wang 2016; Stasulli et al. 2015). Y. pestis also invades macrophages but generates a protective vacuolar compartment, called the Yersinia containing vacuole (YCV), and evades the normal phagosomal maturation pathway and killing (Pujol et al. 2009). This strong but delayed proinflammatory phase associated with massive release of cytokines occurs too late to control the infection, resulting in bacterial invasion, massive tissue destruction, and rapid death. In the bubonic form, bacterial multiplication occurs at the injection site during the initial anti-inflammatory phase, and remains confined for variable time, before colonizing the draining lymph node (Nham et al. 2012). As the host proinflammatory response develops, bacteria disseminate to the liver and spleen which ensure initial bacterial filtering and early blood clearance. However, their capacity is rapidly saturated and massive replication of the bacteria results in septicemia and death (Lawrenz 2010; Sebbane et al. 2005). Animal models Animal models of bubonic and pulmonary plague have been developed in various species, including mice, rats, guinea pigs, and non-human primates, although the mouse is currently the most widely used animal model for plague research (Lawrenz 2010). Experimental studies have used fully virulent Yersinia pestis strains (such as C092), or attenuated strains lacking one or more bacterial virulence factors. In vivo imaging using bacterial strains carrying a bioluminescent reporter has allowed to delineate the successive steps and kinetics of bacterial dissemination (Nham et al. 2012; Sha et al. 2013). Models of pneumonic plague have been produced in the mouse by intranasal (Lathem et al. 2005) or aerosol (Agar et al. 2008) inoculation and result in an infection similar to human pneumonic plague. Upon intranasal infection with strain CO92, mice did not show clinical signs of disease during the first 24 h. Thereafter, they began to hunch over, crowd together, and became inactive. Most of them were

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dead by 3 to 4 days after infection (Lathem et al. 2005). The same observations have been made on inbred and outbred genetic backgrounds (Bubeck et al. 2007). Aerosolization of Y. pestis C092 in a whole-body Madison chamber resulted essentially in the same progression of the disease, with the death of animals in 72–96 h in mice (Agar et al. 2008) and in rats (Agar et al. 2009). Cynomolgus macaques and African green monkeys can be infected by aerosolization to study pneumonic plague and evaluate vaccine efficiency (Lawrenz 2010). Like rodents, they develop a disease closely resembling human pneumonic plague. Mice and rats have also been used to study bubonic plague upon subcutaneous or intradermal injection. Both routes mimic transmission from infected fleas, although intradermal inoculations result in faster kinetics of infection (Gonzalez et  al. 2015). After inoculation, bacteria translocate to the draining lymph node from where they disseminate to the blood and colonize spleen, liver, and other organs. Death occurs from septicemia in 3 to 5 days following inoculation. In rats, like in guinea pigs, the infected lymph node is enlarged, hemorrhagic, and surrounded by edematous tissue, very similar to the human bubo (Sebbane et al. 2005). By contrast, in mice, lymphadenopathy may not be apparent until later stages of the disease (Lawrenz 2010). Genes/loci identified in animal models While the impact of the virulence factors encoded by chromosomal genes or plasmids of Y. pestis on pathogenesis and disease severity have been investigated in detail (Du and Wang 2016; Li and Yang 2008; Pradel et al. 2014), few reports have described host determinants that may contribute to resistance to Y. pestis infection. In the mouse, engineered mutants carrying loss-of-function mutations in specific genes involved in the innate or the adaptive immune responses have been instrumental in identifying the pathways which are subverted by Y. pestis during infection, or essential for efficient host defense. For example, mice inactivated for the Stat6 gene, a key mediator of type 2 immune responses, have a resulting bias towards the type 1 axis and show high levels of protection after vaccination, compared to Stat4−/− which have impaired type 1 immune responses (Elvin and Williamson 2004). Mice with inactivated Ifnar1 gene, therefore lacking the type I interferon (IFN) receptor, were less sensitive to plague and had more neutrophils in the later stage of infection than wildtype mice, which correlated with protection from lethality (Patel et al. 2012). Type I IFN expression was shown to derive from the recognition of intracellular Y. pestis by host TLR7 using mice lacking the Tlr7 gene (Dhariwala et al. 2017). CXC chemokines are potent neutrophil attractants and are involved in host defense against extracellular pathogens. In mice lacking the CXC chemokine CXCL1 (KC)

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or its receptor CXCR2, pneumonic plague progression was accelerated both in bacterial growth and development of primary bronchopneumonia, and associated with delayed neutrophil recruitment in the lung (Eisele et al. 2011). In the black rat (Rattus rattus), the main reservoir of plague in rural environment, field studies, and experimental infections have shown that rats are highly susceptible to Y. pestis in plague free areas and have evolved a genetically based plague resistance inside plague foci which seems associated with a polymorphism in the CCR5 gene (Tollenaere et al. 2008). Further studies identified 22 loci potentially affected by plague-mediated selection, two of which were associated with the surviving rate of rats in experimental infection (Tollenaere et al. 2011). Several candidate genes were suggested in these regions, including the two IL-1 genes (Tollenaere et al. 2013). While all laboratory strains are essentially susceptible to virulent Y. pestis strains, differences have been observed when the KIM5 bacterial strain, which lacks the pgm virulence factor, was used. C57BL/6J mice were susceptible to KIM5 injected intravenously, unlike 129S2/P2, 129S1/ SvImJ, and 129P3/J mice which were resistant (Congleton et al. 2006; Turner et al. 2009). A major host genetic contributor to resistance was mapped to chromosome 1 close to the Il10 gene, but congenic strains demonstrated that this gene was not involved (Turner et al. 2009). Further studies revealed that this region contains at least two distinct loci, both of which are required for resistance on a susceptible C57BL/6 background. The Slc11a1 gene, which plays a role in resistance to Salmonella Typhimurium and lies in the same region, was ruled out (Tencati and Tapping 2016). BALB/cJ mice were also found to resist intravenous injection of KIM5, unlike closely related BALB/cByJ and BALB/cAnNHsd which were highly susceptible (Turner et al. 2008). An F2 cross between BALB/cJ and C57BL/6J identified a major QTL, named prl1, in the major histocompatibility complex region of chromosome 17. This QTL was confirmed in a backcross and introduced in a congenic strain which showed a level of resistance close to that of BALB/ cJ, indicating that prl1 is responsible for the majority of the resistance observed in BALB/cJ mice (Turner et al. 2008). Resistance to a fully virulent Y. pestis strain has been so far reported only in SEG/Pas, a wild-derived inbred strain of the Mus spretus species, in a model of bubonic plague (Blanchet et al. 2011). Upon subcutaneous injection of Y. pestis, SEG/Pas mice showed earlier clinical signs of infection than C57BL/6J mice but survived. Resistance in SEG/Pas mice was associated with early but transient bacterial invasion, whereas in C57BL/6J mice, it was delayed but continuous until death (Demeure et al. 2012). Upon in vitro exposure to Y. pestis, SEG/Pas macrophages expressed a very strong proinflammatory functional profile beyond the prototypic M1 profile of C57BL/6J, with enhanced resistance to Y.

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pestis-induced apoptosis (Pachulec et al. 2017). An interspecific backcross between SEG/Pas and C57BL/6J identified three QTLs (Yprl1, Yprl2, and Yprl3) acting additively, which explained 70% of the difference in survival rate between the two parents (Blanchet et al. 2011). Congenic strains confirmed two (Yprl2 and Yprl3) of the three QTLs and showed that at least four distinct loci (each locus harboring two subregions) were required for mice to survive (Chevallier et al. 2013). While several genomic regions have been associated with resistance to virulent or attenuated strains of Y. pestis, none of them has been narrowed down to a few candidate genes amenable to functional validation. Similar studies are still lacking for Y. pseudotuberculosis. Differences in susceptibility to Yersinia enterocolitica between BALB/c and C57BL/6 mice (Hancock et al. 1986) have been associated with variations in macrophage transcriptome (van Erp et al. 2006) and a different role of IL-12 (Bohn et al. 1998). Relevance to human disease Animal studies have demonstrated the existence of host genetic factors in resistance to Yersinia pestis infection but the identification of resistance genes is still to be achieved. Direct identification of susceptibility genes in humans is unlikely in the case of plague. Upon human infection, it is vital to undertake immediate antibiotic and supporting therapies, which interfere with the evaluation of natural host defense mechanisms. The insight which has been gained by animal models and transcriptomics studies on key host mechanisms which are targeted by the immunosuppressive factors of Y. pestis, or which are essential in host immune responses, provides candidates on which genetic variations could be explored in human cohorts.

Conclusion The use of animal models for scientific research is essential for making fundamental discoveries. This is particularly the case for the study of infectious diseases, in which animal models have been crucial in extending our understanding of the virulence mechanisms employed by pathogens and the contribution of the host immune response as well as host genetics to disease pathogenesis. Indeed, in vivo modeling of bacterial pathogens has provided us a wealth of knowledge on disease pathogenesis in humans. In the future, new technologies combined with rapidly evolving genomic tools should provide us with further insight into the link between host genomics and infectious disease. Epigenetics, for example, is increasingly being recognized as playing a key role in the pathogenesis of infectious diseases. While still an emerging science, new tools are being developed to

Enterobacteria and host resistance to infection

help understand how epigenetic changes and genetic variation influence disease mechanism. Similarly, the recently developed Collaborative Cross panel of recombinant inbred mouse strains holds great promise in systems genetics research and in evaluating the contribution of host genetics to disease outcome. Lastly, efficient gene-editing technologies such as the CRISPR/Cas9 system could prove invaluable in the identification of important host factors involved in susceptibility or resistance to enteric bacteria. Studies of this nature could ultimately lead to new therapeutic strategies to control and combat disease. Acknowledgements  L.C. and X.M. are grateful to Christian Demeure for useful discussions on Y. pestis pathogenesis and mouse models. Funding for this work was supported by the Canadian Institutes of Health Research grants to S.G. (Grant No. MOP 133580) and to D.M. (Grant No. MOP133700); by Genome Canada and Génome Québec to S.G. and D.M.; by the National Science and Engineering Research Council of Canada (NSERC, Grant No. RGPIN-2016-05587) and the Faculty of Science of the University of Ottawa to F.X.C.V. E.K. was supported by studentships from the Faculty of Medicine, McGill University; A.A. by a scholarship from the King Abdullah Scholarship program supported by the Saudi Arabia Cultural Bureau and N.S. by a studentship from the NSERC CREATE program TECHNOMISE.

Compliance with ethical standards  Conflict of interest  On behalf of all authors, the corresponding author states that there is no conflict of interest.

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