Phytobacter diazotrophicus from Intestine of Caenorhabditis elegans Confers Colonization-Resistance against Bacillus nematocida Using Flagellin (FliC) as an Inhibition Factor

Symbiotic microorganisms in the intestinal tract can influence the general fitness of their hosts and contribute to protecting them against invading pathogens. In this study, we obtained isolate Phytobacter diazotrophicus SCO41 from the gut of free-living nematode Caenorhabditis elegans that displayed strong colonization-resistance against invading biocontrol bacterium Bacillus nematocida B16. The colonization-resistance phenotype was found to be mediated by a 37-kDa extracellular protein that was identified as flagellin (FliC). With the help of genome information, the fliC gene was cloned and heterologously expressed in E. coli. It could be shown that the B. nematocida B16 grows in chains rather than in planktonic form in the presence of FliC. Scanning Electronic Microscopy results showed that protein FliC-treated B16 bacterial cells are thinner and longer than normal cells. Localization experiments confirmed that the protein FliC is localized in both the cytoplasm and the cell membrane of B16 strain, in the latter especially at the position of cell division. ZDOCK analysis showed that FliC could bind with serine/threonine protein kinase, membrane protein insertase YidC and redox membrane protein CydB. It was inferred that FliC interferes with cell division of B. nematocidal B16, therefore inhibiting its colonization of C. elegans intestines in vivo. The isolation of P. diazotrophicus as part of the gut microbiome of C. elegans not only provides interesting insights about the lifestyle of this nitrogen-fixing bacterium, but also reveals how the composition of the natural gut microbiota of nematodes can affect biological control efforts by protecting the host from its natural enemies.

FliW and CsrA govern flagellin (FliC) synthesis and play pleiotropic roles in virulence and physiology of Clostridioides difficile R20291

Clostridioides difficile is a Gram-positive, spore-forming, and toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. In C. difficile, the expression of flagellar genes is coupled to toxin gene regulation and bacterial colonization and virulence. The flagellin FliC is responsible for pleiotropic gene regulation during in vivo infection. However, how fliC expression is regulated is unclear. In Bacillus subtilis, flagellin homeostasis and motility are coregulated by flagellar assembly factor FliW, Flagellin Hag (FliC homolog), and CsrA (Carbon storage regulator A), which is referred to as partner-switching mechanism "FliW-CsrA-Hag". In this study, we characterized FliW and CsrA functions by deleting or overexpressing fliW, csrA, and fliW-csrA in C. difficile R20291. We showed that both fliW deletion or csrA overexpression in R20291, and csrA complementation in R20291ΔWA (fliW-csrA codeletion) dramatically decreased FliC production, however, fliC gene transcription was unaffected. While suppression of fliC translation by csrA overexpression was mostly relieved when fliW was coexpressed, and no significant difference in FliC production was detected when only fliW was complemented in R20291ΔWA. Further, loss of fliW led to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model of C. difficile infection (CDI), while fliW-csrA codeletion decreased toxin production and mortality in vivo. Taken together, these data suggest that CsrA negatively modulates fliC expression and FliW indirectly affects fliC expression through inhibition of CsrA post-transcriptional regulation, which seems similar to the "FliW-CsrA-Hag" switch in B. subtilis. Our data also suggest that "FliW-CsrA-fliC/FliC" can regulate many facets of C. difficile R20291 pathogenicity.

Engineering a Novel Bivalent Oral Vaccine against Enteric Fever

Enteric fever is a major global healthcare issue caused largely by Salmonella enterica serovars Typhi and Paratyphi A. The objective of this study was to develop a novel, bivalent oral vaccine capable of protecting against both serovars. Our approach centred on genetically engineering the attenuated S. Typhi ZH9 strain, which has an excellent safety record in clinical trials, to introduce two S. Paratyphi A immunogenic elements: flagellin H:a and lipopolysaccharide (LPS) O:2. We first replaced the native S. Typhi fliC gene encoding flagellin with the highly homologous fliC gene from S. Paratyphi A using Xer-cise technology. Next, we replaced the S. Typhi rfbE gene encoding tyvelose epimerase with a spacer sequence to enable the sustained expression of O:2 LPS and prevent its conversion to O:9 through tyvelose epimerase activity. The resulting new strain, ZH9PA, incorporated these two genetic changes and exhibited comparable growth kinetics to the parental ZH9 strain. A formulation containing both ZH9 and ZH9PA strains together constitutes a new bivalent vaccine candidate that targets both S. Typhi and S. Paratyphi A antigens to address a major global healthcare gap for enteric fever prophylaxis. This vaccine is now being tested in a Phase I clinical trial (NCT04349553).

Identification of Salmonella typhi contamination by amplification fliC gene in grass-jelly from traditional markets and minimarket in Semarang city

Grass-jelly is one of the most popular plants consumed by people in various forms. Contamination can cause various diseases, one of those is typhoid fever by Salmonella typhi. The purpose of this study was to detect S. typhi in grass-jelly based on molecular detection by amplification of the fliC gene using PCR. Validation was done by culture methods on SSA media and biochemical testing. The fliC gene amplification results in grass-jelly samples (A1, A2, B1, B2, C1, and C3) showed the DNA fragments size of about 1500 bp. Colony and biochemical characters isolate Peterongan were lead to S. typhi, whereas another isolate was another Salmonella spp. Grass-jelly samples from the Peterongan market in Semarang were positively contaminated by S. typhi and isolate from Pedurungan and the minimarket was another Salmonella spp. Molecular-based food testing is fast enough and accurate for detecting types of bacterial contaminants but the amplification of only the fliC gene cannot specific for S. typhi.

Emerging Threat of Salmonella typhimurium in Extraintestinal Infections: Cases from a Tertiary Care Hospital in North India

Purpose: Infections due to invasive non-typhoid salmonella can be dangerous and fatal. The mode of infection and the severity varies from the typhoidal fevers. It is important to find the association between clinical features and the infecting serovar to understand the pathophysiology and course of treatment. Methods: In the present study, extra-intestinal specimens (blood, cerebrospinal fluid and pus) from three patients suffering from septicaemia, meningitis and osteomyelitis were received. Micro-biological and biochemical test for species identification and antibiotic susceptibility was done as per standard protocol. Further, PCR based amplification and sequencing of a portion of the flagellin gene (FliC) was done to confirm the serovar. Results: Salmonella enterica was identified from all the three by microbiological and biochemical examination.The sequence of the Flic gene confirmed the serovar to be S. typhimurium. All the patients were treated successfully for the infection by appropriate antibiotic therapy. Conclusion: The study highlights that serovar Typhimurium is common in invasive non-typhoidal salmonellosis and its pathophysiology and virulence factors expression should be understood in various organ types for better treatment options and outcomes.

New Approaches for Escherichia coli Genotyping

Easy-to-perform, fast, and inexpensive methods of differentiation of Escherichia coli strains beyond the species level are highly required. Herein two new, original tools for genotyping of E. coli isolates are proposed. The first of the developed method, a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) test uses a highly variable fliC gene, encoding the H antigen as a molecular target. The designing of universal pair of primers and selection of the optimal restriction enzyme RsaI was preceded by in silico comparative analysis of the sequences of the genes coding for 53 different serotypes of H-antigen (E. coli flagellin). The target fragments of E. coli genomes for MLST method were selected on the basis of bioinformatics analysis of complete sequences of 16 genomes of E. coli. Initially, seven molecular targets were proposed (seven pairs of primers) and five of them were found useful for effective genotyping of E. coli strains. Both developed methods revealed high differentiation power, and a high genetic diversity of the strains tested was observed. Within the group of 71 strains tested, 29 and 47 clusters were revealed with fliC RFLP-PCR and MLST methods, respectively. Differentiation of the strains with the reference BOX-PCR method revealed 31 different genotypes. The in silico analysis revealed that the discriminatory power of the new MLST method is comparable to the Pasteur and Achtman schemes and is higher than the discriminatory power of the method developed by Clermont. From the epidemiology point of view, the outcomes of our investigation revealed that in most cases, the patients were infected with unique strains, probably from environmental sources. However, some strains isolated from different patients of the wards of pediatrics, internal medicine, and neurology were classified to the same genotype when the results of all three methods were taken into account. It could suggest that they were transferred between the patients.

Host Specificity of Flagellins from Segmented Filamentous Bacteria Affects Their Patterns of Interaction with Mouse Ileal Mucosal Proteins

ABSTRACT Segmented filamentous bacteria (SFB) are known modulators of the mammalian immune system. Currently, the technology for investigating SFB culture in vitro is immature, and as a result, the mechanisms of SFB colonization and immune regulation are not yet fully elucidated. In this study, we investigated the gene diversity and host specificity of SFB flagellin genes. The fliC1 and fliC2 genes are relatively conserved, while the fliC3 and fliC4 genes are more variable, especially at the central and C-terminal regions. Host specificity analysis demonstrated that the fliC1 genes do not cluster together based on the host organism, whereas the fliC3 and fliC4 genes were host specific at the nucleotide and deduced amino acid levels. SFB flagellin protein expression in the ileum mucosa and cecal contents was detected by using fluorescence in situ hybridization (FISH) combined with immunohistochemical (IHC) analysis, immunoblotting, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the purified SFB FliC3 protein originating from both mouse and rat was able to activate Toll-like receptor 5 (TLR5)-linked NF-κB signaling, no host specificity was observed. Interestingly, the patterns of interaction with mouse ileum mucosal proteins were different for mouse FliC3 (mFliC3) and rat FliC3 (rFliC3). Gene Ontology (GO) and KEGG analyses indicated that more adherence-related proteins interacted with mFliC3, while more lysosome- and proteolysis-related proteins interacted with rFliC3. In vitro degradation experiments indicated that the stability of rFliC3 was lower than that of mFliC3 when they were incubated with mouse ileum mucosal proteins. In summary, the gene diversity and host specificity of SFB flagellin genes were investigated, and SFB flagellin expression was detected in gut samples. IMPORTANCE Since SFB genomes contain only one copy of each FliC gene, the diversity of FliC is representative of SFB strain diversity. Currently, little is known regarding the diversity and specificity of members of the group of SFB. The work presented herein demonstrates that select SFB strains, exhibiting unique FliC patterns, are present in a variety of mammalian hosts. SFB fliC genes were found to interact with a number of unique targets, providing further evidence for SFB host selection. Together, this work represents a major advancement in identifying SFB and delineating how members of the group of SFB interact with the host. Future examination of FliC genes will likely enhance our knowledge of intestinal colonization by the gut microbiota.

Cloning, expression and purification of the recombinant FliC from Salmonella enteritidis

FliC protein from Salmonella enteritidis is currently interested due to its immunologic adjuvant property for the novel generation of recombinant vaccines. To produce a source for further researches on the immune effects of FliC, we generated an Escherichia coli based on recombinant vector called pET-fliC which is ligated from fliC gene with NdeI and XhoI double digested pET vectors. The results of expression of recombinant FliC, which was induced by IPTG, were confirmed by SDS-PAGE and Western blot probed with anti-6xHis tag. With the purity above 95 %, this recombinant FliC can be used as a material source for next studies on evaluating the adjuvant property of FliC.