Isolation and Characterization of Cytotoxic, Aggregative Citrobacter freundii

Citrobacter freundii is an infrequent but established cause of diarrhea in humans. However, little is known of its genetic diversity and potential for virulence. We analyzed 26 isolates, including 12 from human diarrheal patients, 2 from human fecal samples of unknown diarrheal status, and 12 from animals, insects, and other sources. Pulsed field gel electrophoresis using XbaI allowed us to divide the 26 isolates into 20 pulse types, while multi-locus sequence typing using 7 housekeeping genes allowed us to divide the 26 isolates into 6 sequence types (STs) with the majority belonging to 4 STs. We analyzed adhesion and cytotoxicity to HEp-2 cells in these 26 strains. All were found to adhere to HEp-2 cells. One strain, CF74, which had been isolated from a goat, showed the strongest aggregative adhesion pattern. Lactate dehydrogenase (LDH) released from HEp-2 cells was evaluated as a measure of cytotoxicity, averaging 7.46%. Strain CF74 induced the highest level of LDH, 24.3%, and caused >50% cell rounding, detachment, and death. We named strain CF74 “cytotoxic and aggregative C. freundii.” Genome sequencing of CF74 revealed that it had acquired 7 genomic islands, including 2 fimbriae islands and a type VI secretion system island, all of which are potential virulence factors. Our results show that aggregative adherence and cytotoxicity play an important role in the pathogenesis of C. freundii

Yersinia enterocolitica palearctica serobiotype O:3/4 - a successful group of emerging zoonotic pathogens

<p>Abstract</p> <p>Background</p> <p>High-pathogenic <it>Y. enterocolitica </it>ssp. <it>enterocolitica </it>caused several human outbreaks in Northern America. In contrast, low pathogenic <it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 is responsible for sporadic cases worldwide with asymptomatic pigs being the main source of infection. Genomes of three <it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 human isolates (including the completely sequenced Y11 German DSMZ type strain) were compared to the high-pathogenic <it>Y. enterocolitica </it>ssp. <it>enterocolitica </it>8081 O:8/1B to address the peculiarities of the O:3/4 group.</p> <p>Results</p> <p>Most high-pathogenicity-associated determinants of <it>Y. enterocolitica </it>ssp. <it>enterocolitica </it>(like the High-Pathogenicity Island, <it>yts1 </it>type 2 and <it>ysa </it>type 3 secretion systems) are absent in <it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 genomes. On the other hand they possess alternative putative virulence and fitness factors, such as a different <it>ysp </it>type 3 secretion system, an RtxA-like and insecticidal toxins, and a N-acetyl-galactosamine (GalNAc) PTS system (<it>aga</it>-operon). Horizontal acquisition of two prophages and a tRNA-Asn-associated GIYep-01 genomic island might also influence the <it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 pathoadaptation. We demonstrated recombination activity of the PhiYep-3 prophage and the GIYep-01 island and the ability of the <it>aga</it>-operon to support the growth of the <it>Y. enterocolitica </it>ssp. <it>enterocolitica </it>O:8/1B on GalNAc.</p> <p>Conclusions</p> <p><it>Y. enterocolitica </it>ssp. <it>palearctica </it>serobiotype O:3/4 experienced a shift to an alternative patchwork of virulence and fitness determinants that might play a significant role in its host pathoadaptation and successful worldwide dissemination.</p

MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices

Evolution of Salmonella enterica Virulence via Point Mutations in the Fimbrial Adhesin

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella

Effective assembly of fimbriae in Escherichia coli depends on the translocation assembly module nanomachine

Outer membrane proteins are essential for Gram-negative bacteria to rapidly adapt to changes in their environment. Intricate remodelling of the outer membrane proteome is critical for bacterial pathogens to survive environmental changes, such as entry into host tissues1,​2,​3. Fimbriae (also known as pili) are appendages that extend up to 2 μm beyond the cell surface to function in adhesion for bacterial pathogens, and are critical for virulence. The best-studied examples of fimbriae are the type 1 and P fimbriae of uropathogenic Escherichia coli, the major causative agent of urinary tract infections in humans. Fimbriae share a common mode of biogenesis, orchestrated by a molecular assembly platform called ‘the usher’ located in the outer membrane. Although the mechanism of pilus biogenesis is well characterized, how the usher itself is assembled at the outer membrane is unclear. Here, we report that a rapid response in usher assembly is crucially dependent on the translocation assembly module. We assayed the assembly reaction for a range of ushers and provide mechanistic insight into the β-barrel assembly pathway that enables the rapid deployment of bacterial fimbriae

Genome-scale metabolic reconstructions of multiple <i>Salmonella</i> strains reveal serovar-specific metabolic traits

Salmonella serovars colonize a wide range of hosts but the underlying genetic determinants remain poorly understood. Here, Seif et al. use a network-based computational analysis to link specific metabolic capabilities with host range and nutritional niche

Host-mediated sugar oxidation promotes post-antibiotic pathogen expansion.

Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion