Cross-feeding between intestinal pathobionts promotes their overgrowth during undernutrition.

Child undernutrition is a global health issue associated with a high burden of infectious disease. Undernourished children display an overabundance of intestinal pathogens and pathobionts, and these bacteria induce enteric dysfunction in undernourished mice; however, the cause of their overgrowth remains poorly defined. Here, we show that disease-inducing human isolates of Enterobacteriaceae and Bacteroidales spp. are capable of multi-species symbiotic cross-feeding, resulting in synergistic growth of a mixed community in vitro. Growth synergy occurs uniquely under malnourished conditions limited in protein and iron: in this context, Bacteroidales spp. liberate diet- and mucin-derived sugars and Enterobacteriaceae spp. enhance the bioavailability of iron. Analysis of human microbiota datasets reveals that Bacteroidaceae and Enterobacteriaceae are strongly correlated in undernourished children, but not in adequately nourished children, consistent with a diet-dependent growth synergy in the human gut. Together these data suggest that dietary cross-feeding fuels the overgrowth of pathobionts in undernutrition.

To be or not to be a pathogen: that is the mucosally relevant question.

The human interface with the microbial world has so far largely been considered through the somewhat restrictive angle of host-pathogen interactions resulting in disease. It has consequently largely ignored the daily symbiosis with the microbiota, an ensemble of symbiotic microorganisms engaged in a commensal, and for some of them mutualistic, interaction. This microbiota heavily populates essential surfaces such as the oral and intestinal cavity, the upper respiratory tract, the vagina, and the skin. Host response to the pathogens is characterized by quick recognition combined with strong innate (i.e., inflammatory) and adaptive immune responses, causing microbial eradication often at the cost of significant tissue damage. Response to the symbiotic microbiota is characterized by a process called tolerance that encompasses a complex integration of microbial recognition and tightly controlled innate (i.e., physiological inflammation) and adaptive immune responses. This dichotomy in host response is critical at the gut mucosal surface that is massively colonized by a diverse population of bacteria. The host is therefore permanently facing the challenge of discriminating among symbiotic and pathogenic bacteria in order to offer an adapted response. This asks the fundamental existential question: "to be or not to be… a pathogen." This review has attempted to consider this question from the host angle. What do host mucosal sensing systems see in the bacteria to which they become exposed to establish proper discrimination? A new facet of medicine resides in the dysfunction of this complex balance that has likely forged the complexity of the immune system.

A function for AAMP in Nod2-mediated NF-kappaB activation.

The WD40 repeat containing angio-associated migratory cell protein (AAMP) was identified as a new binding partner of the human nucleotide-binding domain, leucine rich repeat containing (NLR) family member Nod2 in a yeast two-hybrid screen. Co-immunoprecipitations from human cells verified this interaction and revealed that an internal peptide of AAMP spanning three WD40 domains was sufficient for this interaction. AAMP was found to be ubiquitously expressed in different human cell-lines and exhibited a predominant cytosolic localization in epithelial cells. Functionally, using overexpression and siRNA knock-down, we showed that AAMP modulates Nod2- and Nod1-mediated NF-kappaB activation in HEK293T cells. Taken together, our data support a new function of AAMP in regulating innate immune responses initiated by the NLR protein Nod2.

A newborn mouse model for the study of intestinal pathogenesis of shigellosis.

Shigella infection is characterized by the induction of acute inflammation, which is responsible for the massive tissue destruction of the intestinal mucosa. A murine model would be a valuable tool for gaining a better understanding of the physiopathology of shigellosis and the host immune response to Shigella infection, but adult mice do not develop disease upon oral inoculation. We therefore attempted to develop a model of infection in newborn mice. Four-day-old mice inoculated with 50 microl of 5 x 10(9) invasive wild-type Shigella flexneri 5a were susceptible to bacterial infection, but mice inoculated with the non-invasive strain BS176 were not. Histologically, 4-day-old mice infected with the invasive strain presented intestinal lesions and inflammation similar to those described in patients with shigellosis. Moreover, cytokine and chemokine responses consistent with inflammation were observed. Lower bacterial inocula induced less severe intestinal damage. In contrast, 5-day-old mice inoculated with either the invasive or the non-invasive strain were not infected. We have thus established a mouse model that is suitable for the study of the pathogenesis of intestinal Shigella infection.

Shigellosis: innate mechanisms of inflammatory destruction of the intestinal epithelium, adaptive immune response, and vaccine development.

Acute infectious colitis remains a major pediatric issue of worldwide impact because it still represents a significant public health burden among the larger group of diarrheal diseases with the highest mortality rate. It is also a relevant model of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis. Among cases of acute colitis of infectious origin, shigellosis is certainly the one that has benefited the most from a significant research effort. Shigella, the causative agent, is a Gram-negative bacterium that has the capacity to invade, disrupt, and cause inflammatory destruction of the intestinal epithelial barrier. The molecular and cellular bases of this invasive phenotype essentially encompass crossing of the epithelial lining, apoptotic killing of macrophages, entry into epithelial cells, and escape into the cytoplasm, followed by cell-to-cell spread. Intracellular colonization is likely to protect the micro-organisms from killing by humoral and cellular effectors of the innate immune response. Concurrently, the capacity of Shigella to reprogram invaded epithelial cells to produce proinflammatory mediators plays a major role in the strong inflammatory profile of the disease. This profile is likely to impact on the nature and quality of the adaptive response, which is dominated by humoral protection at the mucosal level.

Host-pathogen interactions: the seduction of molecular cross talk.

Bacterial pathogens have evolved two major strategies to colonise the intestinal epithelium. Adherent microorganisms bind to the apical pole of the intestinal epithelium, whereas invasive microorganisms disrupt and invade the epithelium. Recognition of the genetic bases of bacterial pathogenicity and analysis of the molecular cross talks established between pathogens and their mammalian target cells have illuminated this diversity of interactions. We have compared the strategies of enteroinvasive pathogens, with emphasis on bacterial species such as Shigella, Yersinia, and Salmonella, that represent paradigms of interaction. Cross talks leading to alteration of the epithelial cell actin cytoskeleton appear as a recurrent theme during entry and dissemination into epithelial cells. Other cross talks alter the trafficking of cellular vesicles and induce changes in the intracellular compartment in which they reside, thus creating niches favourable to bacterial survival and growth. Finally, a variety of strategies also exist to deal with other components of the epithelial barrier, such as macrophages. Pro-phagocytic, anti-phagocytic, and pro-apoptotic processes appear to be of particular importance.

Characterization of the interaction partners of secreted proteins and chaperones of Shigella flexneri.

The type III secretion (TTS) system of Gram-negative pathogenic bacteria is composed of proteins that assemble into the TTS machinery, proteins that are secreted by this machinery and specific chaperones that are required for storage and sometimes secretion of these proteins. Many sequential protein interactions are involved in the TTS pathway to deliver effector proteins to host cells. We used the yeast two-hybrid system to investigate the interaction partners of the Shigella flexneri effectors and chaperones. Libraries of preys containing random fusions with fragments of the TTS proteins were screened using effectors and chaperones as baits. Interactions between the effectors IpaB and IpaC and their chaperone IpgC were detected by this method, and interaction domains were identified. Using a His-tagged IpgC protein to co-purify truncated IpaB and IpaC proteins, we showed that the chaperone-binding domain was unique and located in the N-terminus of these proteins. This domain was not required for the secretion of recombinant proteins but was involved in the stability of IpaC and instability of IpaB. Homotypic interactions were identified with the baits IpaA, IpaB and IpaC. Interactions between effectors and components of the TTS machinery were also selected that might give insights into regulation of the TTS process.

Blockade of CD14 aggravates experimental shigellosis.

Shigella infections lead to severe inflammation associated with destruction of colonic mucosa. We assessed the effect of in vivo blockade of CD14 on the outcome of experimental Shigella infection in rabbits. A total of 17 rabbits were divided into two groups: 8 received a single i.v. dose of anti-rabbit CD14 monoclonal antibody prior to infection with an invasive Shigella flexneri strain; the remainder served as controls. The anti-CD14-treated rabbits exhibited more severe tissue destruction and a 50-fold increase in bacterial invasion of the intestinal mucosa when compared to controls. Similar numbers of polymorphonuclear leukocytes were recruited to the intestinal mucosa in both groups despite the massive bacterial invasion seen in the CD14-blocked group. No statistically significant differences were seen in levels of IL-1beta nor in the ratio of IL-1RA/IL-1beta for either group. In contrast, higher quantities of TNF-alpha were observed in the CD14-blocked group. To conclude, anti-CD14 treatment had a detrimental effect on the capacity of Shigella-infected animals to clear the infection.

Phagocytosis of bacterial pathogens: implications in the host response.

Phagocytosis of bacterial pathogens is at the heart of the pathogenesis of infections. Pathogens have evolved a large array of strategies to escape the deleterious effect of phagocytosis by professional phagocytes among which avoiding phagocytosis, killing the phagocytes or surviving inside them are the most 'popular' solutions. Bacterial pathogens are also using induction of phagocytic entry into non-professional phagocytic cells, such as epithelial cells, as a strategy of survival and multiplication. We have taken enteroinvasive micro-organisms such as Yersinia, Shigella and Salmonella as a paradigm of the significance of phagocytosis/antiphagocytosis in the development of an infection and on the elicitation of the host response.

Innate immune responses of epithelial cells following infection with bacterial pathogens.

The ability to discriminate between pathogenic and non-pathogenic bacteria is extremely important for epithelial cells lining mucosal surfaces and is particularly so in colonic epithelial cells. Accumulating evidence suggests that bacterial recognition systems used by epithelial cells are very different from those in cells of the myeloid lineage and are likely to have developed to maintain mucosal surfaces in a state of homeostasis with the normal microbial flora. Bacterial invasion of epithelial cells or breach of the epithelial barrier provides a signal to epithelial cells to initiate inflammatory responses, which are key events for the clearance of the infecting microbe. Therefore, elucidation of the mechanisms by which epithelial cells recognize bacteria and bacterial products, and of the nature of the innate immune responses that are triggered by these factors are important for our understanding of both the immunology of mucosal surfaces and bacterial pathogenesis.

Predominance of serotype-specific mucosal antibody response in Shigella flexneri-infected humans living in an area of endemicity.

The mucosal humoral immune response elicited following Shigella flexneri infection in patients living in Antananarivo districts (Madagascar Island) was evaluated by measuring the gut-derived, circulating immunoglobulin A (IgA) antibody-secreting cells (ASC) specific for the major bacterial antigen lipopolysaccharide (LPS). Fifty, 34, 11, and 5% of the S. flexneri-positive patients were infected with serotypes 2a, 1a, 4a, and 3a, respectively. The total number of IgA ASC in infected patients increased significantly, compared to the number in healthy controls, early after the onset of disease. The number of anti-homologous LPS IgA ASC varied among individuals and peaked between days 5 and 10 after the onset of the disease. In the S. flexneri 1a- and 2a-infected patients, the level of IgA ASC cross-reactivity to heterologous S. flexneri serotypes was weak. These data indicate that S. flexneri 2a and 1a are the predominant strains responsible for shigellosis in this area of endemicity and that the anti-LPS antibody response following natural infection is mainly directed against serotype-specific determinants.

CARD4/Nod1 mediates NF-kappaB and JNK activation by invasive Shigella flexneri.

Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF-kappaB and c-Jun N-terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll-like receptors (TLRs), which mediate LPS responsiveness from the outside-in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance-like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF-kappaB induction. Dominant-negative versions of CARD4 block activation of NF-kappaB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease-resistance proteins.

Parameters underlying successful protection with live attenuated mutants in experimental shigellosis.

Because the use of live attenuated mutants of Shigella spp. represents a promising approach to protection against bacillary dysentery (M. E. Etherridge, A. T. M. Shamsul Hoque, and D. A. Sack, Lab. Anim. Sci. 46:61-66, 1996), it becomes essential to rationalize this approach in animal models in order to optimize attenuation of virulence in the vaccine candidates, as well as their route and mode of administration, and to define the correlates of protection. In this study, we have compared three strains of Shigella flexneri 5--the wild-type M90T, an aroC mutant, and a double purE aroC mutant--for their pathogenicity, immunogenicity, and protective capacity. Protection against keratoconjunctivitis, induced by wild-type M90T, was used as the protection read out in guinea pigs that were inoculated either intranasally or intragastrically. Following intranasal immunization, the aroC mutant elicited weak nasal tissue destruction compared to M90T and achieved protection correlated with high levels of local anti-lipopolysaccharide immunoglobulin A (IgA), whereas the purE aroC double mutant, which also elicited weak tissue destruction, was not protective and elicited a low IgA response. Conversely, following intragastric immunization, only the M90T purE aroC double mutant elicited protection compared to both the aroC mutant and the wild-type strain. This mutant caused mild inflammatory destruction, particularly at the level of Peyer's patches, but it persisted much longer within the tissues. This could represent an essential parameter of the protective response that, in this case, did not clearly correlate with high anti-lipopolysaccharide IgA titers.

Microbes and microbial toxins: paradigms for microbial-mucosal interactions III. Shigellosis: from symptoms to molecular pathogenesis.

Interaction of Shigella flexneri with epithelial cells includes contact of bacteria with the cell surface and release of Ipa proteins through a specialized type III secreton. A complex signaling process involving activation of small GTPases of the Rho family and c-src causes major rearrangements of the subcortical cytoskeleton, thereby allowing bacterial entry by macropinocytosis. After entry, shigellae escape to the cell cytoplasm and initiate intracytoplasmic movement through polar nucleation and assembly of actin filaments caused by bacterial surface protein IcsA, which binds and activates neuronal Wiskoff-Aldrich syndrome protein (N-WASP), thus inducing actin nucleation in an Arp 2/3-dependent mechanism. Actin-driven motility promotes efficient colonization of the host cell cytoplasm and rapid cell-to-cell spread via protrusions that are engulfed by adjacent cells in a cadherin-dependent process. Bacterial invasion turns infected cells to strongly proinflammatory cells through sustained activation of nuclear factor-kappaB. A major consequence is interleukin (IL)-8 production, which attracts polymorphonuclear leukocytes (PMNs). On transmigration, PMNs disrupt the permeability of this epithelium and promote its invasion by shigellae. At the early stage of infection, M cells of the follicle-associated epithelium allow bacterial translocation. Subsequent apoptotic killing of macrophages in a caspase 1-dependent process causes the release of IL-1beta and IL-18, which accounts for the initial steps of inflammation.

Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton.

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram-negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the "needle complex" (NC), produced an average image at 17 A resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2-3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N-terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.

The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri.

Bacteria of Shigella spp. are the causative agents of shigellosis. The virulence traits of these pathogens include their ability to enter into epithelial cells and induce apoptosis in macrophages. Expression of these functions requires the Mxi-Spa type III secretion apparatus and the secreted IpaA-D proteins, all of which are encoded by a virulence plasmid. In wild-type strains, the activity of the secretion apparatus is tightly regulated and induced upon contact of bacteria with epithelial cells. To investigate the repertoire of proteins secreted by Shigella flexneri in conditions of active secretion, we determined the N-terminal sequence of 14 proteins that are secreted by a mutant in which secretion was deregulated. Sequencing of the virulence plasmid pWR100 of the S. flexneri strain M90T (serotype 5) has allowed us to identify the genes encoding these secreted proteins and suggests that approximately 25 proteins are secreted by the type III secretion apparatus. Analysis of the G+C content and the relative positions of genes and open reading frames carried by the plasmid, together with information concerning the localization and function of encoded proteins, suggests that pWR100 contains blocks of genes of various origins, some of which were initially carried by four different plasmids.