Salmonella T3SS effector SseK1 arginine-glycosylates the two-component response regulator OmpR to alter bile salt resistance.

Type III secretion system (T3SS) effector proteins are primarily recognized for binding host proteins to subvert host immune response during infection. Besides their known host target proteins, several T3SS effectors also interact with endogenous bacterial proteins. Here we demonstrate that the Salmonella T3SS effector glycosyltransferase SseK1 glycosylates the bacterial two-component response regulator OmpR on two arginine residues, R15 and R122. Arg-glycosylation of OmpR results in reduced expression of ompF, a major outer membrane porin gene. Glycosylated OmpR has reduced affinity to the ompF promoter region, as compared to the unglycosylated form of OmpR. Additionally, the Salmonella ΔsseK1 mutant strain had higher bile salt resistance and increased capacity to form biofilms, as compared to WT Salmonella, thus linking OmpR glycosylation to several important aspects of bacterial physiology.

Arginine glycosylation regulates UDP-GlcNAc biosynthesis in Salmonella enterica.

The Salmonella enterica SseK1 protein is a type three secretion system effector that glycosylates host proteins during infection on specific arginine residues with N-acetyl glucosamine (GlcNAc). SseK1 also Arg-glycosylates endogenous bacterial proteins and we thus hypothesized that SseK1 activities might be integrated with regulating the intrabacterial abundance of UPD-GlcNAc, the sugar-nucleotide donor used by this effector. After searching for new SseK1 substrates, we found that SseK1 glycosylates arginine residues in the dual repressor-activator protein NagC, leading to increased DNA-binding affinity and enhanced expression of the NagC-regulated genes glmU and glmS. SseK1 also glycosylates arginine residues in GlmR, a protein that enhances GlmS activity. This Arg-glycosylation improves the ability of GlmR to enhance GlmS activity. We also discovered that NagC is a direct activator of glmR expression. Salmonella lacking SseK1 produce significantly reduced amounts of UDP-GlcNAc as compared with Salmonella expressing SseK1. Overall, we conclude that SseK1 up-regulates UDP-GlcNAc synthesis both by enhancing the DNA-binding activity of NagC and by increasing GlmS activity through GlmR glycosylation. Such regulatory activities may have evolved to maintain sufficient levels of UDP-GlcNAc for both bacterial cell wall precursors and for SseK1 to modify other bacterial and host targets in response to environmental changes and during infection.

Bacterial community analysis of purulent material from liver abscesses of crossbred cattle and Holstein steers fed finishing diets with or without tylosin.

Liver abscesses in feedlot cattle are polymicrobial infections. Culture-based studies have identified Fusobacterium necrophorum as the primary causative agent, but a number of other bacterial species are frequently isolated. The incidence of liver abscesses is highly variable and is affected by a number of factors, including cattle type. Holstein steers raised for beef production have a higher incidence than crossbred feedlot cattle. Tylosin is the commonly used antimicrobial feed additive to reduce the incidence of liver abscesses. The objective of this study was to utilize 16S ribosomal RNA amplicon sequence analyses to analyze the bacterial community composition of purulent material of liver abscesses of crossbred cattle (n = 24) and Holstein steers (n = 24), each fed finishing diet with or without tylosin. DNA was extracted and the V3 and V4 regions of the 16S rRNA gene were amplified, sequenced, and analyzed. The minimum, mean, and maximum sequence reads per sample were 996, 177,070, and 877,770, respectively, across all the liver abscess samples. Sequence analyses identified 5 phyla, 14 families, 98 genera, and 102 amplicon sequence variants (ASV) in the 4 treatment groups. The dominant phyla identified were Fusobacteria (52% of total reads) and Proteobacteria (33%). Of the top 25 genera identified, 17 genera were Gram negative and 8 were Gram positive. The top 3 genera, which accounted for 75% of the total reads, in the order of abundance, were Fusobacterium, Pseudomonas, and Bacteroides. The relative abundance, expressed as percent of total reads, of phyla, family, and genera did not differ (P > 0.05) between the 4 treatment groups. Generic richness and evenness, determined by Shannon-Weiner and Simpson's diversity indices, respectively, did not differ between the groups. The UniFrac distance matrices data revealed no clustering of the ASV indicating variance between the samples within each treatment group. Co-occurrence network analysis at the genus level indicated a strong association of Fusobacterium with 15 other genera, and not all of them have been previously isolated from liver abscesses. In conclusion, the culture-independent method identified the bacterial composition of liver abscesses as predominantly Gram negative and Fusobacterium as the dominant genus, followed by Pseudomonas. The bacterial community composition did not differ between crossbred and Holstein steers fed finishing diets with or without tylosin.

An intra-bacterial activity for a T3SS effector.

Many Gram-negative bacterial pathogens interact with mammalian cells by using type III secretion systems (T3SS) to inject virulence proteins into host cells. A subset of these injected protein 'effectors' are enzymes that inhibit the function of host proteins by catalyzing the addition of unusual post-translational modifications. The E. coli and Citrobacter rodentium NleB effectors, as well as the Salmonella enterica SseK effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected, due to the activity of chaperones that keep the effectors in a structural state permissive for secretion. While performing mass spectrometry experiments to identify glycosylation substrates of NleB orthologs, we unexpectedly observed that the bacterial glutathione synthetase (GshB) is glycosylated by NleB on arginine residue R256. NleB-mediated glycosylation of GshB resulted in enhanced GshB activity, leading to an increase in glutathione production, and promoted C. rodentium survival in oxidative stress conditions. These data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that arginine-GlcNAcylation, once thought to be restricted to host cell compartments, also plays an important role in regulating bacterial physiology.

Structural basis for arginine glycosylation of host substrates by bacterial effector proteins.

The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. Here we report that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face SNi mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens.

Immunization With Skp Delivered on Outer Membrane Vesicles Protects Mice Against Enterotoxigenic Escherichia coli Challenge.

Outer membrane vesicles (OMVs) are promising vaccine components because they combine antigen and adjuvant in a single formulation. Detoxified Salmonella enterica strains that express penta-acylated lipid A retain OMV immunogenicity but with reduced reactogenicity. We have previously shown that a recombinant form of the enterotoxigenic Escherichia coli (ETEC) 17 kilodalton protein (Skp) protects mice in a pulmonary challenge model, when fused to the glutathione-S-transferase (GST) epitope and combined with cholera toxin. Here we compared directly the efficacy of expressing Skp in detoxified Salmonella OMVs to GST-Skp for their ability to protect mice against ETEC challenge. We observed that the display of Skp on OMVs, in the absence of exogenous adjuvant, protects the mice as well as the recombinant GST-Skp with adjuvant, showing that we can achieve protection when antigen and adjuvant are administered as a single formulation. Collectively, these data demonstrate the utility of using OMVs for the expression and display of antigens for use in vaccine development and validate previously published work demonstrating that immunization with Skp is efficacious in protecting mice against ETEC challenge.

Immunization with the MipA, Skp, or ETEC_2479 Antigens Confers Protection against Enterotoxigenic E. coli Strains Expressing Different Colonization Factors in a Mouse Pulmonary Challenge Model.

Achieving cross-protective efficacy against multiple bacterial strains or serotypes is an important goal of vaccine design. Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in underdeveloped nations. We have been interested in identifying and characterizing ETEC antigens that generate protective immune responses independent of ETEC colonization factor (CF) expression. Our previous studies used proteomics to identify the ETEC MipA, Skp, and ETEC_2479 proteins as effective in protecting mice from homologous challenge with ETEC H10407 using a pulmonary inoculation model. This model permits analysis of mouse survival, bacterial clearance, and the production of secretory IgA (sIgA) and has been employed previously for studies of enteric pathogens for which robust oral challenge models do not exist. MipA belongs to a family of proteins involved in remodeling peptidoglycan. Skp rescues misdirected outer membrane proteins. ETEC_2479 is predicted to function as an outer membrane porin. These proteins are conserved in pathogenic ETEC strains as well as in commensal Proteobacteria. Antibodies produced against the ETEC MipA, Skp, and ETEC_2479 proteins also reduced the adherence of multiple ETEC strains differing in CF type to intestinal epithelial cells. Here we characterized the ability of 10 heterologous ETEC strains that differ in CF type to cause clinical signs of illness in mice after pulmonary challenge. ETEC strains C350C1A, E24377A, E7476A, WS2173A, and PE360 caused variable degrees of lethality in this mouse model, while ETEC strains B7A, WS6866B, 2230, ARG-2, and 8786 did not. Subsequent challenge experiments in which mice were first vaccinated intranasally with MipA, Skp, or ETEC_2479, when combined with cholera toxin, showed both that each antigen was protective and that protection was strongly correlated with fecal IgA concentrations. We conclude that the MipA, Skp, or ETEC_2479 antigens generate protection in the mouse pulmonary challenge model against ETEC strains that express different CFs.

Variable-number tandem-repeat analysis of leptospiral DNA isolated from canine urine samples molecularly confirmed to contain pathogenic leptospires.

OBJECTIVE To use variable-number tandem-repeat (VNTR) analysis to determine the infecting serovar and strain for leptospiral DNA isolated from canine urine samples confirmed through PCR testing to contain pathogenic leptospires and to evaluate the sensitivity and specificity of microscopic agglutination testing (MAT) for identifying the infecting serogroup. DESIGN Diagnostic survey and test evaluation. SAMPLE Leptospiral DNA isolated from urine samples from 98 dogs confirmed through PCR testing to have pathogenic leptospires in their urine. PROCEDURES VNTR analysis of DNA isolates was performed to identify the infecting leptospiral serovar and strain by use of primer pairs for the loci 4, 7, 10, and Lb5. Eighteen pathogenic and 2 saprophytic leptospiral serovars were used as reference strains for VNTR analysis. Results of MAT were compared with those of the PCR assay and VNTR analysis to determine the sensitivity and specificity of MAT for diagnosing leptospirosis and identifying the infecting serovar at various reciprocal titers. RESULTS VNTR analysis identified Leptospira kirschneri serovar Grippotyphosa strain DF as the most common infecting serovar in dogs (78/98 [80%]). Thirteen unique VNTR patterns could not be identified by comparison with the Leptospira reference strains used. The MAT had a maximum sensitivity of 41% and a specificity of 100% for identifying Grippotyphosa as the infecting serogroup. CONCLUSIONS AND CLINICAL RELEVANCE Findings confirmed the importance of Leptospira serovar Grippotyphosa among dogs in the United States. Serologic testing had poor sensitivity for identifying the infecting serogroup, and conclusions about emerging serogroups should be cautiously interpreted when serologic data are reported.

Vaccinating with conserved Escherichia coli antigens does not alter the mouse intestinal microbiome.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) causes diarrheal disease. Antigenic and structural heterogeneity among ETEC colonization factors has complicated vaccine development efforts. Identifying and characterizing conserved ETEC antigens that induce protective immunity is therefore of interest. We previously characterized three proteins (MipA, Skp, and ETEC_2479) that protected mice in an intranasal ETEC challenge model after vaccination. However, these proteins are conserved not only in multiple ETEC isolates, but also in commensal bacteria. While the impact of inactivated viral vaccines and live-attenuated bacterial vaccines on the host microbiota have been examined, the potential impact of using subunit vaccines consisting of antigens that are also encoded by commensal organisms has not been investigated. FINDINGS: We addressed this issue by characterizing changes to mouse intestinal microbiomes as a function of vaccination. We failed to observe significant changes to mouse health, to mouse weight gain as a function of time, or to the diversity or richness of mouse intestinal microbiomes, as measured by analyzing alpha- and beta-diversity, as well as overall community structure, before and after vaccination. CONCLUSIONS: We conclude that despite the conservation of MipA, Skp, and ETEC_2479 among Gram-negative bacteria, vaccination with these antigens fails to alter significantly the host intestinal microbiome.

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.

Detection of Escherichia coli O157:H7 in cattle feces using a polymerase chain reaction-based fluorogenic 5' nuclease (TaqMan) detection assay after secondary enrichment.

Currently, methods for recovering and identifying Escherichia coli O157:H7 from cattle feces are inconsistent and hindered by their inability to specifically and rapidly detect small numbers of organisms from this complex and highly variable matrix. A standard approach for isolating and characterizing E. coli O157:H7 from cattle feces was compared with a polymerase chain reaction (PCR)-based 5' nuclease assay specific for E. coli O157:H7 that included a secondary enrichment step. The PCR-based method proved a better indicator of the presence of the organism than the culture procedure. Retests indicated that the inclusion of a secondary enrichment step and the subsequent analysis by the 5' nuclease assay were reproducible and specific. Escherichia coli O157:H7 could be detected in fecal samples that were otherwise negative after a primary enrichment step, immunomagnetic separation, and plating onto sorbitol MacConkey agar plates containing cefixime and tellurite (CT-SMAC). In samples that were initially identified as culture positive but PCR negative, retesting of the culture isolates on CT-SMAC indicated that the sorbitol fermentation interpretations could frequently not be repeated in retests, whereas retesting using the 5' nuclease assay on the original samples demonstrated a high level of agreement with the initial PCR conclusions. These results indicate the necessity of confirmatory evaluation of isolates culturally recovered by standard cultural methods that involve the interpretation of CT-SMAC. The high level of disagreement between initial culture results and retests, and the high level of agreement between initial PCR results and retests, indicates the advantages of a gene-based detection system for identifying E. coli O157:H7 in cattle feces. Screening large numbers of fecal samples for E. coli O157:H7 would appear to be feasible by integrating the use of enrichment media in serial rounds of incubation with a PCR-based fluorogenic detection procedure in high throughput detection systems that had automated liquid-handling capabilities.