PmrC (EptA) and CptA Negatively Affect Outer Membrane Vesicle Production in Citrobacter rodentium.

Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-type Citrobacter rodentium, the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline. C. rodentium uses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, the C. rodentium ΔpmrAB strain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription of pmrC (also known as eptA) and cptA OMV production in strains lacking either pmrC (eptA) or cptA was similarly increased in comparison to that of the wild type. Importantly, plasmid complementation of C. rodentium strains with either pmrC (eptA) or cptA resulted in a drastic inhibition of OMV production. Finally, we showed that ß-lactamase and CroP, two enzymes found in the C. rodentium periplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by which C. rodentium and possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genes pmrC (eptA) and cptAIMPORTANCE Although OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogen Citrobacter rodentium In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron in C. rodentium These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria.

The bacterial arginine glycosyltransferase effector NleB preferentially modifies Fas-associated death domain protein (FADD).

The inhibition of host innate immunity pathways is essential for the persistence of attaching and effacing pathogens such as enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium during mammalian infections. To subvert these pathways and suppress the antimicrobial response, attaching and effacing pathogens use type III secretion systems to introduce effectors targeting key signaling pathways in host cells. One such effector is the arginine glycosyltransferase NleB1 (NleBCR in C. rodentium) that modifies conserved arginine residues in death domain-containing host proteins with N-acetylglucosamine (GlcNAc), thereby blocking extrinsic apoptosis signaling. Ectopically expressed NleB1 modifies the host proteins Fas-associated via death domain (FADD), TNFRSF1A-associated via death domain (TRADD), and receptor-interacting serine/threonine protein kinase 1 (RIPK1). However, the full repertoire of arginine GlcNAcylation induced by pathogen-delivered NleB1 is unknown. Using an affinity proteomic approach for measuring arginine-GlcNAcylated glycopeptides, we assessed the global profile of arginine GlcNAcylation during ectopic expression of NleB1, EPEC infection in vitro, or C. rodentium infection in vivo NleB overexpression resulted in arginine GlcNAcylation of multiple host proteins. However, NleB delivery during EPEC and C. rodentium infection caused rapid and preferential modification of Arg117 in FADD. This FADD modification was extremely stable and insensitive to physiological temperatures, glycosidases, or host cell degradation. Despite its stability and effect on the inhibition of apoptosis, arginine GlcNAcylation did not elicit any proteomic changes, even in response to prolonged NleB1 expression. We conclude that, at normal levels of expression during bacterial infection, NleB1/NleBCR antagonizes death receptor-induced apoptosis of infected cells by modifying FADD in an irreversible manner.

NleB/SseK effectors from Citrobacter rodentium, Escherichia coli, and Salmonella enterica display distinct differences in host substrate specificity.

Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells. Some of these effectors are enzymes that modify host proteins to subvert their normal functions. NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium Moreover, Salmonella enterica strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3. However, there are conflicting reports regarding the activities and host protein targets among the NleB/SseK orthologs. Therefore, here we performed in vitro glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and Crodentium NleB blocked TNF-mediated NF-κB pathway activation, whereas SseK2 and NleB2 did not. C. rodentium NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. C. rodentium NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein). SseK3 and NleB2 were not active against either substrate. We also found that EHEC NleB1 glycosylated two GAPDH arginine residues, Arg197 and Arg200, and that these two residues were essential for GAPDH-mediated activation of TNF receptor-associated factor 2 ubiquitination. These results provide evidence that members of this highly conserved family of bacterial virulence effectors target different host protein substrates and exhibit distinct cellular modes of action to suppress host responses.

Citrobacter rodentium NleB Protein Inhibits Tumor Necrosis Factor (TNF) Receptor-associated Factor 3 (TRAF3) Ubiquitination to Reduce Host Type I Interferon Production.

Interferon signaling plays important roles in both intestinal homeostasis and in the host response to pathogen infection. The extent to which bacterial pathogens inhibit this host pathway is an understudied area of investigation. We characterized Citrobacter rodentium strains bearing deletions in individual type III secretion system effector genes to determine whether this pathogen inhibits the host type I IFN response and which effector is responsible. The NleB effector limited host IFN-ß production by inhibiting Lys(63)-linked ubiquitination of TNF receptor-associated factor 3 (TRAF3). Inhibition was dependent on the glycosyltransferase activity of NleB. GAPDH, a target of NleB during infection, bound to TRAF3 and was required for maximal TRAF3 ubiquitination. NleB glycosyltransferase activity inhibited GAPDH-TRAF3 binding, resulting in reduced TRAF3 ubiquitination. Collectively, our data reveal important interplay between GAPDH and TRAF3 and suggest a mechanism by which the NleB effector inhibits type I IFN signaling.

Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity.

UNLABELLED: Bacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagic Escherichia coli (EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP, found in the murine pathogen Citrobacter rodentium, which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the abilities of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded LL-37 and CRAMP at similar rates. In contrast, C. rodentium CroP cleaved CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and CRAMP were independent of the bacterial background and substrate sequence specificity, as OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was α-helical and CRAMP was unstructured under our experimental conditions. By altering the α-helicity of LL-37 and CRAMP, we found that decreasing LL-37 α-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP α-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins of C. rodentium and E. coli. In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments. IMPORTANCE: Omptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer membrane, their active site facing the extracellular environment, and their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here, we describe subtle differences between the substrate specificities of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases.

The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host.

Bacterial pathogens produce a number of autotransporters that possess diverse functions. These include the family of serine protease autotransporters of Enterobacteriaceae (SPATEs) produced by enteric pathogens such as Shigella flexneri and enteroaggregative Escherichia coli. Of these SPATEs, one termed "protein involved in colonization," or Pic, has been shown to possess mucinase activity in vitro, but to date, its role in in vivo enteric pathogenesis is unknown. Testing a pic null (ΔpicC) mutant in Citrobacter rodentium, a natural mouse pathogen, found that the C. rodentium ΔpicC strain was impaired in its ability to degrade mucin in vitro compared to the wild type. Upon infection of mice, the ΔpicC mutant exhibited a hypervirulent phenotype with dramatically heavier pathogen burdens found in intestinal crypts. ΔpicC mutant-infected mice suffered greater barrier disruption and more severe colitis and weight loss, necessitating their euthanization between 10 and 14 days postinfection. Notably, the virulence of the ΔpicC mutant was normalized when the picC gene was restored; however, a PicC point mutant causing loss of mucinase activity did not replicate the ΔpicC phenotype. Exploring other aspects of PicC function, the ΔpicC mutant was found to aggregate to higher levels in vivo than wild-type C. rodentium. Moreover, unlike the wild type, the C. rodentium ΔpicC mutant had a red, dry, and rough (RDAR) morphology in vitro and showed increased activation of the innate receptor Toll-like receptor 2 (TLR2). Interestingly, the C. rodentium ΔpicC mutant caused a degree of pathology similar to that of wild-type C. rodentium when infecting TLR2-deficient mice, showing that despite its mucinase activity, PicC's major role in vivo may be to limit C. rodentium's stimulation of the host's innate immune system.

Inhibition of outer membrane proteases of the omptin family by aprotinin.

Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.

An iron-containing dodecameric heptosyltransferase family modifies bacterial autotransporters in pathogenesis.

Autotransporters deliver virulence factors to the bacterial surface by translocating an effector passenger domain through a membrane-anchored barrel structure. Although passenger domains are diverse, those found in enteric bacteria autotransporters, including AIDA-I in diffusely adhering Escherichia coli (DAEC) and TibA in enterotoxigenic E. coli, are commonly glycosylated. We show that AIDA-I is heptosylated within the bacterial cytoplasm by autotransporter adhesin heptosyltransferase (AAH) and its paralogue AAH2. AIDA-I heptosylation determines DAEC adhesion to host cells. AAH/AAH2 define a bacterial autotransporter heptosyltransferase (BAHT) family that contains ferric ion and adopts a dodecamer assembly. Structural analyses of the heptosylated TibA passenger domain reveal 35 heptose conjugates forming patterned and solenoid-like arrays on the surface of a ß helix. Additionally, CARC, the AIDA-like autotransporter from Citrobacter rodentium, is essential for colonization in mice and requires heptosylation by its cognate BAHT. Our study establishes a bacterial glycosylation system that regulates virulence and is essential for pathogenesis.

NleB, a bacterial effector with glycosyltransferase activity, targets GAPDH function to inhibit NF-κB activation.

Modulation of NF-κB-dependent responses is critical to the success of attaching/effacing (A/E) human pathogenic E. coli (EPEC and EHEC) and the natural mouse pathogen Citrobacter rodentium. NleB, a highly conserved type III secretion system effector of A/E pathogens, suppresses NF-κB activation, but the underlying mechanisms are unknown. We identified the mammalian glycolysis enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an NleB-interacting protein. Further, we discovered that GAPDH interacts with the TNF receptor-associated factor 2 (TRAF2), a protein required for TNF-α-mediated NF-κB activation, and regulates TRAF2 polyubiquitination. During infection, NleB functions as a translocated N-acetyl-D-glucosamine (O-GlcNAc) transferase that modifies GAPDH. NleB-mediated GAPDH O-GlcNAcylation disrupts the TRAF2-GAPDH interaction to suppress TRAF2 polyubiquitination and NF-κB activation. Eliminating NleB O-GlcNAcylation activity attenuates C. rodentium colonization of mice. These data identify GAPDH as a TRAF2 signaling cofactor and reveal a virulence strategy employed by A/E pathogens to inhibit NF-κB-dependent host innate immune responses.

A structural motif is the recognition site for a new family of bacterial protein O-glycosyltransferases.

The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDA-associated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA-I, which comprises imperfect repeats of a 19-amino-acid consensus sequence and is predicted to form a ß-helix. Here, we show that Aah homologues can be found in many Gram-negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA-like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19-amino-acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of ß-helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a ß-helical polypeptide lacking the 19-amino-acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif.

An outer membrane protease of the omptin family prevents activation of the Citrobacter rodentium PhoPQ two-component system by antimicrobial peptides.

The PhoPQ two-component system of the intracellular pathogen Salmonella enterica senses and controls resistance to alpha-helical antimicrobial peptides (AMPs) by regulating covalent modifications of lipid A. A homologue of the phoPQ operon was found in the genome of the murine enteric extracellular pathogen, Citrobacter rodentium. Here we report that C. rodentium PhoPQ was apparently unable to mediate activation of target genes in the presence of alpha-helical AMPs. However, these AMPs activated C. rodentium PhoPQ expressed in a S. entericaDeltaphoPQ mutant. Analysis of the outer membrane (OM) fractions of the C. rodentium wild-type and DeltaphoPQ strains led to the identification of an omptin family protease (CroP) that was absent in DeltaphoPQ. Deletion of croP in C. rodentium resulted in higher susceptibility to alpha-helical AMPs, indicating a direct role of CroP in AMP resistance. CroP greatly contributed to the protection of the OM from AMP damage by actively degrading alpha-helical AMPs before they reach the periplasmic space. Accordingly, transcriptional activation of PhoP-regulated genes by alpha-helical AMPs was restored in the DeltacroP mutant. This study shows that resistance to alpha-helical AMPs by the extracellular pathogen C. rodentium relies primarily on the CroP OM protease.