The CpxRA two-component system of adherent and invasive Escherichia coli contributes to epithelial cell invasion and early-stage intestinal fitness in a dysbiotic mouse model mediated by type 1 fimbriae expression.

Adherent and invasive Escherichia coli (AIEC) is a pathobiont that is involved in the onset and exacerbation of Crohn's disease. Although the inducible expression of virulence traits is a critical step for AIEC colonization in the host, the mechanism underlying AIEC colonization remains largely unclear. We here showed that the two-component signal transduction system CpxRA contributes to AIEC gut competitive colonization by activating type 1 fimbriae expression. CpxRA from AIEC strain LF82 functioned as a transcriptional regulator, as evidenced by our finding that an isogenic cpxRA mutant exhibits reduced expression of cpxP, a known regulon gene. Transcription levels of cpxP in LF82 increased in response to envelope stress, such as exposure to antimicrobials compromising the bacterial membrane, whereas the cpxRA mutant did not exhibit this response. Furthermore, we found that the cpxRA mutant exhibits less invasiveness into host cells than LF82, primarily due to reduced expression of the type 1 fimbriae. Finally, we found that the cpxRA mutant is impaired in gut competitive colonization in a mouse model. The colonization defects were reversed by the introduction of a plasmid encoding the cpxRA gene or expressing the type 1 fimbriae. Our findings indicate that modulating CpxRA activity could be a promising approach to regulating AIEC-involved Crohn's disease.

Possible link between colonization of the gastrointestinal tract by Citrobacter rodentium in C57BL/6 mice and microbiota composition.

Colonization resistance, conferred by the host's microbiota through both direct and indirect protective actions, serves to protect the host from enteric infections. Here, we identified the specific members of the gut microbiota that impact gastrointestinal colonization by Citrobacter rodentium, a murine pathogen causing colonic crypt hyperplasia. The gut colonization levels of C. rodentium in C57BL/6 mice varied among breeding facilities, probably due to differences in microbiota composition. A comprehensive analysis of the microbiota revealed that specific members of the microbiota may influence gut colonization by C. rodentium, thus providing a potential link between the two.

Fatty Acid Homeostasis Tunes Flagellar Motility by Activating Phase 2 Flagellin Expression, Contributing to Salmonella Gut Colonization.

Long-chain-fatty-acid (LCFA) metabolism is a fundamental cellular process in bacteria that is involved in lipid homeostasis, energy production, and infection. However, the role of LCFA metabolism in Salmonella enterica serovar Typhimurium (S. Tm) gut infection remains unclear. Here, using a murine gastroenteritis infection model, we demonstrate involvement of LCFA metabolism in S. Tm gut colonization. The LCFA metabolism-associated transcriptional regulator FadR contributes to S. Tm gut colonization. fadR deletion alters the gene expression profile and leads to aberrant flagellar motility of S. Tm. Colonization defects in the fadR mutant are attributable to altered swimming behavior characterized by less frequently smooth swimming, resulting from reduced expression of the phase 2 flagellin FljB. Notably, changes in lipid LCFA composition by fadR deletion lead to reduced expression of fljB, which is restored by exogenous LCFA. Therefore, LCFA homeostasis may maintain proper flagellar motility by activating fljB expression, contributing to S. Tm gut colonization. Our findings improve the understanding of the effect of luminal LCFA on the virulence of enteric pathogens.

O-acetylesterase activity of Bifidobacterium bifidum sialidase facilities the liberation of sialic acid and encourages the proliferation of sialic acid scavenging Bifidobacterium breve.

Bifidobacterium bifidum possesses two extracellular sialidases (SiaBb1 and SiaBb2) that release free sialic acid from mucin sialoglycans, which can be utilized via cross-feeding by Bifidobacterium breve that, otherwise, is prevented from utilizing this nutrient source. Modification of sialic acids with O-acetyl esters is known to protect mucin glycans from degradation by bacterial sialidases. Compared to SiaBb2, SiaBb1 has an additional O-acetylesterase (Est) domain. We aimed to elucidate the role of the SiaBb1 Est domain from B. bifidum in sialic acid cross-feeding within Bifidobacterium. Pre-treatment of mucin secreted from bovine submaxillary glands (BSM) using His6 -tagged-Est and -SiaBb2 released a higher amount of sialic acid compared to the pre-treatment by His6 -SiaBb2. Growth of B. breve increased with an increase in nanE expression when supplemented with both His6 -Est- and His6 -SiaBb2-treated BSM. These results indicate that the esterase activity of the SiaBb1 Est domain enhances the efficiency of SiaBb2 to cleave sialic acid from mucin. This free sialic acid can be utilized by coexisting sialic acid scavenging B. breve via cross-feeding. Here, we provide the molecular mechanism underlying the unique sialoglycan degradation property of B. bifidum which is mediated by the complementary activities of SiaBb1 and SiaBb2 in the context of sialic acid cross-feeding.

uvrY Deletion and Acetate Reduce Gut Colonization of Crohn's Disease-Associated Adherent-Invasive Escherichia coli by Decreasing Expression of Type 1 Fimbriae.

Adherent-invasive Escherichia coli (AIEC) is involved in onset and/or exacerbation of Crohn's disease (CD). AIEC adapts to the gut environment by altering gene expression programs, leading to successful gut-lumen colonization. However, the underlying mechanism of gut colonization is still far from clarified. Here, we show the role of UvrY, a response regulator of bacterial two-component signal transduction systems, in AIEC gut colonization. An AIEC mutant lacking the uvrY gene exhibited impairment of competitive colonization in the murine intestinal tract. UvrY contributes to functional expression of type 1 fimbriae by activating expression of small RNA CsrB, which confers adherence and invasion into epithelial cells on AIEC. In contrast, acetate suppresses the UvrY-dependent expression of type 1 fimbriae, resulting in less efficient cell invasion and attenuated gut colonization. Our findings might lead to therapeutic interventions for CD, in which inhibitions of UvrY activation and acetate supplementation reduce the colonization levels of AIEC by decreasing type 1 fimbria expression.

A Salmonella type III effector, PipA, works in a different manner than the PipA family effectors GogA and GtgA.

Nuclear factor-kappa B (NF-κB) plays a critical role in the host defense against microbial pathogens. Many pathogens modulate NF-κB signaling to establish infection in their host. Salmonella enterica serovar Typhimurium (S. Typhimurium) possesses two type III secretion systems (T3SS-1 and T3SS-2) and directly injects many effector proteins into host cells. It has been reported that some effectors block NF-κB signaling, but the molecular mechanism of the inactivation of NF-κB signaling in S. Typhimurium is poorly understood. Here, we identified seven type III effectors-GogA, GtgA, PipA, SseK1, SseK2, SseK3, and SteE-that inhibited NF-κB activation in HeLa cells stimulated with TNF-α. We also determined that only GogA and GtgA are involved in regulation of the activation of NF-κB in HeLa cells infected with S. Typhimurium. GogA, GtgA, and PipA are highly homologous to one another and have the consensus zinc metalloprotease HEXXH motif. Our experiments demonstrated that GogA, GtgA, and PipA each directly cleaved NF-κB p65, whereas GogA and GtgA, but not PipA, inhibited the NF-κB activation in HeLa cells infected with S. Typhimurium. Further, expressions of the gogA or gtgA gene were induced under the SPI-1-and SPI-2-inducing conditions, but expression of the pipA gene was induced only under the SPI-2-inducing condition. We also showed that PipA was secreted into RAW264.7 cells through T3SS-2. Finally, we indicated that PipA elicits bacterial dissemination in the systemic stage of infection of S. Typhimurium via a T3SS-1-independent mechanism. Collectively, our results suggest that PipA, GogA and GtgA contribute to S. Typhimurium pathogenesis in different ways.

Gut Microbiota-Derived Propionate Regulates the Expression of Reg3 Mucosal Lectins and Ameliorates Experimental Colitis in Mice.

BACKGROUND AND AIMS: Regenerating islet-derived protein type 3 [Reg3] lectins are antimicrobial peptides at mucosal surfaces of the gut, whose expression is regulated by pathogenic gut microbes via interleukin-22- or Toll-like receptor signalling. In addition to antimicrobial effects, tissue protection is hypothesized, but has been poorly investigated in the gut. METHODS: We applied antibiotic-induced microbiota perturbations, gnotobiotic approaches and a dextran-sodium sulfate [DSS] colitis model to assess microbial Reg3 regulation in the intestines and its role in colitis. We also used an intestinal organoid model to investigate this axis in vitro. RESULTS: First, we studied whether gut commensals are involved in Reg3 expression in mice, and found that antibiotic-mediated reduction of Clostridia downregulated intestinal Reg3B. A loss in Clostridia was accompanied by a significant reduction of short-chain fatty acids [SCFAs], and knock-out [KO] mice for SCFA receptors GPR43 and GPR109 expressed less intestinal Reg3B/-G. Propionate was found to induce Reg3 in intestinal organoids and in gnotobiotic mice colonized with a defined, SCFA-producing microbiota. Investigating the role of Reg3B as a protective factor in colitis, we found that Reg3B-KO mice display increased inflammation and less crypt proliferation in the DSS colitis model. Propionate decreased colitis and increased proliferation. Treatment of organoids exposed to DSS with Reg3B or propionate reversed the chemical injury with a loss of expression of the stem-cell marker Lgr5 and Olfm4. CONCLUSIONS: Our results suggest that Clostridia can regulate Reg3-associated epithelial homeostasis through propionate signalling. We also provide evidence that the Reg3-propionate axis may be an important mediator of gut epithelial regeneration in colitis.

A Peptidoglycan Amidase Activator Impacts Salmonella enterica Serovar Typhimurium Gut Infection.

Salmonella enterica serovar Typhimurium is an important foodborne pathogen that causes diarrhea. S. Typhimurium elicits inflammatory responses and colonizes the gut lumen by outcompeting the microbiota. Although evidence is accumulating with regard to the underlying mechanism, the infectious stage has not been adequately defined. Peptidoglycan amidases are widely distributed among bacteria and play a prominent role in peptidoglycan maintenance by hydrolyzing peptidoglycans. Amidase activation is required for the regulation of at least one of two cognate activators, NlpD or EnvC (also called YibP). Recent studies established that the peptidoglycan amidase AmiC-mediated cell division specifically confers a fitness advantage on S Typhimurium in the inflamed gut. However, it remains unknown which cognate activators are involved in the amidase activation and how the activators influence Salmonella sp. pathogenesis. Here, we characterize the role of two activators, NlpD and EnvC, in S Typhimurium cell division and gut infection. EnvC was found to contribute to cell division of S Typhimurium cells through the activation of AmiA and AmiC. The envC mutant exhibited impairments in gut infection, including a gut colonization defect and reduced ability to elicit inflammatory responses. Importantly, the colonization defect of the envC mutant was unrelated to the microbiota but was conferred by attenuated motility and chemotaxis of S Typhimurium cells, which were not observed in the amiA amiC mutant. Furthermore, the envC mutant was impaired in its induction of mucosal inflammation and sustained gut colonization. Collectively, our findings provide a novel insight into the peptidoglycan amidase/cognate activator circuits and their dependent pathogenesis.

Twin-Arginine Translocation System Is Involved in Citrobacter rodentium Fitness in the Intestinal Tract.

The twin-arginine translocation (Tat) system is involved in not only a wide array of cellular processes but also pathogenesis in many bacterial pathogens; thus, this system is expected to become a novel therapeutic target to treat infections. To the best of our knowledge, involvement of the Tat system has not been reported in the gut infection caused by Citrobacter rodentium Here, we studied the role of Tat in C. rodentium gut infection, which resembles human infection with enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). A C. rodentium Tat loss-of-function mutant displayed prolonged gut colonization, which was explained by reduced inflammatory responses and, particularly, neutrophil infiltration. Further, the Tat mutant had colonization defects upon coinfection with the wild-type strain of C. rodentium The Tat mutant also became hypersensitive to bile acids, and an increase in fecal bile acids fostered C. rodentium clearance from the gut lumen. Finally, we show that the chain form of C. rodentium cells, induced by a Tat-dependent cell division defect, exhibits impaired resistance to bile acids. Our findings indicate that the Tat system is involved in gut colonization by C. rodentium, which is associated with neutrophil infiltration and resistance to bile acids. Interventions that target the Tat system, as well as luminal bile acids, might thus be promising therapeutic strategies to treat human EHEC and EPEC infections.

Salmonella enterica Effectors SifA, SpvB, SseF, SseJ, and SteA Contribute to Type III Secretion System 1-Independent Inflammation in a Streptomycin-Pretreated Mouse Model of Colitis.

Salmonella enterica serovar Typhimurium (S. Typhimurium) induces inflammatory changes in the ceca of streptomycin-pretreated mice. In this mouse model of colitis, the type III secretion system 1 (T3SS-1) has been shown to induce rapid inflammatory change in the cecum at early points, 10 to 24 h after infection. Five proteins, SipA, SopA, SopB, SopD, and SopE2, have been identified as effectors involved in eliciting intestinal inflammation within this time range. In contrast, a T3SS-1-deficient strain was shown to exhibit inflammatory changes in the cecum at 72 to 120 h postinfection. However, the effectors eliciting T3SS-1-independent inflammation remain to be clarified. In this study, we focused on two T3SS-2 phenotypes, macrophage proliferation and cytotoxicity, to identify the T3SS-2 effectors involved in T3SS-1-independent inflammation. We identified a mutant strain that could not induce cytotoxicity in a macrophage-like cell line and that reduced intestinal inflammation in streptomycin-pretreated mice. We also identified five T3SS-2 effectors, SifA, SpvB, SseF, SseJ, and SteA, associated with T3SS-1-independent macrophage cytotoxicity. We then constructed a strain lacking T3SS-1 and all the five T3SS-2 effectors, termed T1S5. The S. Typhimurium T1S5 strain significantly reduced cytotoxicity in macrophages in the same manner as a mutant invA spiB strain (T1T2). Finally, the T1S5 strain elicited no inflammatory changes in the ceca of streptomycin-pretreated mice. We conclude that these five T3SS-2 effectors contribute to T3SS-1-independent inflammation.

Tat-exported peptidoglycan amidase-dependent cell division contributes to Salmonella Typhimurium fitness in the inflamed gut.

Salmonella enterica serovar Typhimurium (S. Tm) is a cause of food poisoning accompanied with gut inflammation. Although mucosal inflammation is generally thought to be protective against bacterial infection, S. Tm exploits the inflammation to compete with commensal microbiota, thereby growing up to high densities in the gut lumen and colonizing the gut continuously at high levels. However, the molecular mechanisms underlying the beneficial effect of gut inflammation on S. Tm competitive growth are poorly understood. Notably, the twin-arginine translocation (Tat) system, which enables the transport of folded proteins outside bacterial cytoplasm, is well conserved among many bacterial pathogens, with Tat substrates including virulence factors and virulence-associated proteins. Here, we show that Tat and Tat-exported peptidoglycan amidase, AmiA- and AmiC-dependent cell division contributes to S. Tm competitive fitness advantage in the inflamed gut. S. Tm tatC or amiA amiC mutants feature a gut colonization defect, wherein they display a chain form of cells. The chains are attributable to a cell division defect of these mutants and occur in inflamed but not in normal gut. We demonstrate that attenuated resistance to bile acids confers the colonization defect on the S. Tm amiA amiC mutant. In particular, S. Tm cell chains are highly sensitive to bile acids as compared to single or paired cells. Furthermore, we show that growth media containing high concentrations of NaCl and sublethal concentrations of antimicrobial peptides induce the S. Tm amiA amiC mutant chain form, suggesting that gut luminal conditions such as high osmolarity and the presence of antimicrobial peptides impose AmiA- and AmiC-dependent cell division on S. Tm. Together, our data indicate that Tat and the Tat-exported amidases, AmiA and AmiC, are required for S. Tm luminal fitness in the inflamed gut, suggesting that these proteins might comprise effective targets for novel antibacterial agents against infectious diarrhea.

Salmonella enterica Serovar Typhimurium CpxRA Two-Component System Contributes to Gut Colonization in Salmonella-Induced Colitis.

Salmonella enterica, a common cause of diarrhea, has to colonize the gut lumen to elicit disease. In the gut, the pathogen encounters a vast array of environmental stresses that cause perturbations in the bacterial envelope. The CpxRA two-component system monitors envelope perturbations and responds by altering the bacterial gene expression profile. This allows Salmonella to survive under such harmful conditions. Therefore, CpxRA activation is likely to contribute to Salmonella gut infection. However, the role of the CpxRA-mediated envelope stress response in Salmonella-induced diarrhea is unclear. Here, we show that CpxRA is dispensable for the induction of colitis by S. enterica serovar Typhimurium, whereas it is required for gut colonization. We prove that CpxRA is expressed during gut infection and that the presence of antimicrobial peptides in growth media activates the expression of CpxRA-regulated genes. In addition, we demonstrate that a S Typhimurium strain lacking the cpxRA gene is able to cause colitis but is unable to continuously colonize the gut. Finally, we show that CpxRA-dependent gut colonization requires the host gut inflammatory response, while DegP, a CpxRA-regulated protease, is dispensable. Our findings reveal that the CpxRA-mediated envelope stress response plays a crucial role in Salmonella gut infection, suggesting that CpxRA might be a promising therapeutic target for infectious diarrhea.

Inflammatory bactericidal lectin RegIIIß: Friend or foe for the host?

In the inflamed gut, the bactericidal lectin RegIIIß is massively produced by intestinal mucosa. RegIIIß binds peptidoglycan and lipid A respectively, and thus can kill certain Gram-positive and Gram-negative bacteria, including the gut commensal microbiota and enteropathogenic bacteria. Considering the expression pattern and bactericidal activity, RegIIIß is believed to be a host defense factor for protecting against the infection with enteropathogenic bacteria. However, it was poorly understood how RegIIIß recognizes the target bacteria and kill them, and how RegIIIß plays role(s) in infectious diarrhea. Therefore, our recent study has focused on RegIIIß-target recognition, killing of Gram-negative bacteria, and host protective functions of RegIIIß for infectious diarrhea inflicted by Salmonella Typhimurium. Here, we discuss novel insights into the protective role of RegIIIß in infectious diarrhea, and propose avenues towards novel therapeutic interventions for Salmonella diarrhea.

Salmonella Typhimurium PagP- and UgtL-dependent resistance to antimicrobial peptides contributes to the gut colonization.

Mucosal barrier formed by cationic antimicrobial peptides (CAMPs) is believed to be crucial for host protection from pathogenic gut infection. However, some pathogens can develop resistance to the CAMPs to survive in hosts. Salmonella enterica is a common cause of acute diarrhea. During the course of this disease, the pathogen must continuously colonize the gut lumen, which contains CAMPs. However, it is incompletely understood whether the resistance of Salmonella strains to CAMPs contributes to the development of gut infections. PhoPQ two-component system-dependent lipid A modifications confer resistance to CAMPs in S. enterica serovar Typhimurium. Therefore, we introduced mutations into the PhoPQ-regulated genes in an S. Typhimurium strain, obtaining pagP ugtL and pmrA mutant strains. Each mutant strain demonstrated a distinct spectrum of the resistance to CAMPs. Using streptomycin mouse model for Salmonella diarrhea, we show that the pagP ugtL, but not pmrA, mutant strain had a gut colonization defect. Furthermore, the pagP ugtL, but not pmrA, mutant strain had decreased outer membrane integrity and susceptibility to magainin 2, an alpha-helical CAMP. Taken together, the PagP- and UgtL-dependent resistance to CAMPs was demonstrated to contribute to sustained colonization in the gut. This may be due to the robust outer membrane of S. Typhimurium, inducing the resistance to alpha-helical CAMPs such as α-defensins. Our findings indicate that the development of resistance to CAMPs is required for the S. Typhimurium gut infection.

The Bactericidal Lectin RegIIIß Prolongs Gut Colonization and Enteropathy in the Streptomycin Mouse Model for Salmonella Diarrhea.

The bactericidal lectin RegIIIß is inducibly produced by intestinal epithelial cells as a defense against infection by enteropathogens. In the gut lumen, RegIIIß kills not only certain enteropathogens, but also some commensal bacteria; thus, RegIIIß is also thought to be an innate immune effector shaping microbiota composition and establishing intestinal homeostasis. Using the streptomycin mouse model for Salmonella colitis, we show that RegIIIß can promote sustained gut colonization of Salmonella Typhimurium and prolong enteropathy. RegIIIß expression was associated with suppression of Bacteroides spp. in the gut lumen, prolonged disease-associated alterations in colonic metabolism, and reduced luminal vitamin B6 levels. Supplementation with Bacteroides spp. or vitamin B6 accelerated pathogen clearance from the gut and remission of enteropathy. Our findings indicate that interventions at the level of RegIIIß and supplementation with Bacteroides spp. or vitamin B6 might open new avenues for therapeutic intervention in the context of Salmonella colitis.

Chromobacterium haemolyticum Pneumonia Possibly Due to the Aspiration of Runoff Water.

Here, we report a case of pneumonia possibly caused by the aspiration of runoff water containing Chromobacterium haemolyticum. A 69-year-old man became intoxicated with alcohol, fell into a ditch, and lost consciousness after suffering a blow to the head. The lower half of his body was completely paralyzed because of damage to his spinal cord, and he aspirated runoff water from the ditch. Chest computed tomography scans revealed consolidation in the right upper lobe and bilateral lower lobes. A sputum culture detected gram-negative bacteria that was identified as C. haemolyticum. Antibacterial chemotherapy was initiated, and the clinical course was favorable. To the best of our knowledge, this is the first report of C. haemolyticum pneumonia in the literature.

[Virulence determinant of Chromobacterium violaceum].

Chromobacterium violaceum is a Gram-negative bacterium that infects humans and animals with fatal sepsis. The infection with C. violaceum is rare in case of those who are healthy, but once established, C. violaceum causes sever disease accompanied by abscess formation in the lungs, liver and spleen. Furthermore, C. violaceum is resistant to a broad range of antibiotics, which in some cases renders the antimicrobial therapy for this infection difficult. Thus, the infection with C. violaceum displays high mortality rates unless initial proper antimicrobial therapy. In contrast, the infection mechanism had completely remained unknown. To this end, we have tried to identify virulence factors-associated with C. violaceum infection. Two distinct type III secretion systems (TTSSs) were thought to be one of the most important virulence factors, which are encoded by Chromobacterium pathogenicity island 1/1a and 2 (Cpi-1/-1a and -2) respectively. Our results have shown that Cpi-1/-1a-encoded TTSS, but not Cpi-2, is indispensable for the virulence in a mouse infection model. C. violaceum caused fulminant hepatitis in a Cpi-1/-1a-encoded TTSS-dependent manner. We next have identified 16 novel effectors secreted from Cpi-1/-1a-encoded TTS machinery. From these effectors, we found that CopE (Chromobacterium outer protein E) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases. CopE acts as GEF for Rac1 and Cdc42, leading to induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades cultured human epithelial cells in a CopE-dependent manner. Finally, an inactivation of CopE by disruption of copE gene or amino acid point mutation leading to loss of GEF activity attenuates significantly the mouse virulence of C. violaceum. These results suggest that Cpi-1/-1a-encoded TTSS is a major virulence determinant for C. violaceum infection, and that CopE contributes to the virulence in part of this pathogen.

Draft Genome Sequence of Chromobacterium haemolyticum Causing Human Bacteremia Infection in Japan.

Chromobacterium haemolyticum is a Gram-negative bacterium displaying remarkable hemolysis against human and sheep erythrocytes. In addition, C. haemolyticum infects humans, in which the infection mechanism remains unknown. We report here the draft genome sequence of C. haemolyticum strain T124, isolated from a young patient with sepsis in Japan.

Functional characterization of the type III secretion ATPase SsaN encoded by Salmonella pathogenicity island 2.

A type III secretion system (T3SS) is utilized by a large number of gram-negative bacteria to deliver effectors directly into the cytosol of eukaryotic host cells. One essential component of a T3SS is an ATPase that catalyzes the unfolding of proteins, which is followed by the translocation of effectors through an injectisome. Here we demonstrate a functional role of the ATPase SsaN, a component of Salmonella pathogenicity island 2 T3SS (T3SS-2) in Salmonella enterica serovar Typhimurium. SsaN hydrolyzed ATP in vitro and was essential for T3SS function and Salmonella virulence in vivo. Protein-protein interaction analyses revealed that SsaN interacted with SsaK and SsaQ to form the C ring complex. SsaN and its complex co-localized to the membrane fraction under T3SS-2 inducing conditions. In addition, SsaN bound to Salmonella pathogenicity island 2 (SPI-2) specific chaperones, including SsaE, SseA, SscA, and SscB that facilitated translocator/effector secretion. Using an in vitro chaperone release assay, we demonstrated that SsaN dissociated a chaperone-effector complex, SsaE and SseB, in an ATP-dependent manner. Effector release was dependent on a conserved arginine residue at position 192 of SsaN, and this was essential for its enzymatic activity. These results strongly suggest that the T3SS-2-associated ATPase SsaN contributes to T3SS-2 effector translocation efficiency.

Outer membrane permeabilization is an essential step in the killing of gram-negative bacteria by the lectin RegIIIß.

The C-type lectin RegIIIß can kill certain Gram-positive and Gram-negative bacteria. The susceptibility of S. Typhimurium depends on the bacterial growth phase, i.e., bacteria from the logarithmic growth phase do bind RegIIIß and are subsequently killed. Lipid A is one of the bacterial targets for RegIIIß. However, at the molecular level, it is not understood how RegIIIß interacts with and kills Gram-negative bacteria. Here, we show that RegIIIß interacts with Gram-negative bacteria in two distinct steps. Initially, it binds to surface-exposed lipid A. The lipid A can be shielded by the O-antigen of lipopolysaccharide (LPS), as indicated by the exquisite susceptibility of wbaP mutants to RegIIIß-mediated killing. Increased cell viability after incubation with an anti-lipid A antibody also supports this conclusion. This RegIIIß-binding permeabilizes the outer membrane to hydrophobic dyes like Ethidium bromide or to bulky bacteriolytic enzymes like lysozyme. Conversely, compromising the outer membrane integrity by the mild detergent Triton X-100 enhances the antibacterial effect of RegIIIß. Based on our observations, we conclude that RegIIIß interacts with Gram-negative bacteria in two subsequent steps. Initially, it binds to the outer membrane thus leading to outer membrane permeabilization. This initial step is necessary for RegIIIß to reach a second, still not well understood target site (presumably localized in the periplasm or the cytoplasmic membrane), thereby triggering bacterial death. This provides novel insights into the outer membrane-step of the bactericidal mechanism of RegIIIß.