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

FimH Adhesin of Type 1 Fimbriae Is a Potent Inducer of Innate Antimicrobial Responses Which Requires TLR4 and Type 1 Interferon Signalling

Components of bacteria have been shown to induce innate antiviral immunity via Toll-like receptors (TLRs). We have recently shown that FimH, the adhesin portion of type 1 fimbria, can induce the innate immune system via TLR4. Here we report that FimH induces potent in vitro and in vivo innate antimicrobial responses. FimH induced an innate antiviral state in murine macrophage and primary MEFs which was correlated with IFN-β production. Moreover, FimH induced the innate antiviral responses in cells from wild type, but not from MyD88−/−, Trif−/−, IFN−α/βR−/− or IRF3−/− mice. Vaginal delivery of FimH, but not LPS, completely protected wild type, but not MyD88−/−, IFN-α/βR−/−, IRF3−/− or TLR4−/− mice from subsequent genital HSV-2 challenge. The FimH-induced innate antiviral immunity correlated with the production of IFN-β, but not IFN-α or IFN-γ. To examine whether FimH plays a role in innate immune induction in the context of a natural infection, the innate immune responses to wild type uropathogenic E. coli (UPEC) and a FimH null mutant were examined in the urinary tract of C57Bl/6 (B6) mice and TLR4-deficient mice. While UPEC expressing FimH induced a robust polymorphonuclear response in B6, but not TLR4−/− mice, mutant bacteria lacking FimH did not. In addition, the presence of TLR4 was essential for innate control of and protection against UPEC. Our results demonstrate that FimH is a potent inducer of innate antimicrobial responses and signals differently, from that of LPS, via TLR4 at mucosal surfaces. Our studies suggest that FimH can potentially be used as an innate microbicide against mucosal pathogens

A three-way comparative genomic analysis of Mannheimia haemolytica isolates

<p>Abstract</p> <p>Background</p> <p><it>Mannhemia haemolytica </it>is a Gram-negative bacterium and the principal etiological agent associated with bovine respiratory disease complex. They transform from a benign commensal to a deadly pathogen, during stress such as viral infection and transportation to feedlots and cause acute pleuropneumonia commonly known as shipping fever. The U.S beef industry alone loses more than one billion dollars annually due to shipping fever. Despite its enormous economic importance there are no specific and accurate genetic markers, which will aid in understanding the pathogenesis and epidemiology of <it>M. haemolytica </it>at molecular level and assist in devising an effective control strategy.</p> <p>Description</p> <p>During our comparative genomic sequence analysis of three <it>Mannheimia haemolytica </it>isolates, we identified a number of genes that are unique to each strain. These genes are "high value targets" for future studies that attempt to correlate the variable gene pool with phenotype. We also identified a number of high confidence single nucleotide polymorphisms (hcSNPs) spread throughout the genome and focused on non-synonymous SNPs in known virulence genes. These SNPs will be used to design new hcSNP arrays to study variation across strains, and will potentially aid in understanding gene regulation and the mode of action of various virulence factors.</p> <p>Conclusions</p> <p>During our analysis we identified previously unknown possible type III secretion effector proteins, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated sequences (Cas). The presence of CRISPR regions is indicative of likely co-evolution with an associated phage. If proven functional, the presence of a type III secretion system in <it>M. haemolytica </it>will help us re-evaluate our approach to study host-pathogen interactions. We also identified various adhesins containing immuno-dominant domains, which may interfere with host-innate immunity and which could potentially serve as effective vaccine candidates.</p

Bacteriophage Cocktail and Microcin-Producing Probiotic Escherichia coli Protect Mice Against Gut Colonization With Multidrug-Resistant Escherichia coli Sequence Type 131

Non-antibiotic measures are needed to reduce the rate of infections due to multidrug-resistant organisms (MDROs), including by eliminating the commensal reservoir that underlies such strains’ dissemination and leads to recurrent infections. Here, we tested a cocktail of pre-selected bacteriophages and an engineered microcin C7-producing probiotic Escherichia coli Nissle-1917 strain for their ability to reduce gut colonization by an E. coli strain from sequence type 131 (ST131)-H30R, which is the major clonal group of MDROs among extraintestinal clinical E. coli isolates. Although the bacteriophage cocktail was highly effective against ST131-H30R strains both in vitro and in a murine model of subcutaneous sepsis, it was only weakly and transiently effective against gut colonization by the target ST131-H30R strain (0.5 log10 decrease on d + 1: p &amp;lt; 0.001; no significant effect on d + 4 and beyond). The probiotic strain, while also highly active against ST131-H30R in vitro, was ineffective against ST131-H30R gut colonization despite its abundant presence in feces. Nonetheless, despite failing as decolonizing agents when administered separately, when co-administered the bacteriophage cocktail and probiotic strain exhibited striking synergy against ST131-H30R gut colonization. This combinatory effect was most pronounced on d + 1 (3.3 log10 target strain decrease: p &amp;lt; 0.001), and persisted until d + 7 (0.5 log10 decrease; p &amp;lt; 0.02.). Although by d + 10 the ST131-H30R load was fully restored, these findings provide proof of concept for combined bacteriophage-plus-probiotic administration to reduce or, possibly, to prevent gut colonization with MDROs in high-risk individuals.</jats:p

Exercise-induced apoptosis in rat kidney is mediated by both angiotensin II AT1 and AT2 receptors

Histol Histopathol. 2006 May;21(5):459-66. Exercise-induced apoptosis in rat kidney is mediated by both angiotensin II AT1 and AT2 receptors. Podhorska-Okolow M, Dziegiel P, Gomulkiewicz A, Kisiela D, Dolinska-Krajewska B, Jethon Z, Carraro U, Zabel M. Source Department of Histology and Embryology, Medical University of Wroclaw, Wroclaw, Poland. [email protected] Abstract Excessive physical exercise may lead to disturbance of the entire homeostasis in the body, including damage not only in skeletal muscles but also in many distant organs. The mechanisms responsible for the exercise-induced changes could include oxidative stress or angiotensin II. We previously showed that acute exercise led to apoptosis in kidney but not as a result of oxidative stress. In this study, we examined the role of angiotensin II and its AT1 and AT2 receptors in mediation of exercise-induced apoptosis in kidney. We clearly demonstrated that acute physical exercise induced apoptosis in renal cells of distal convoluted tubuli and cortical and medullary collecting ducts. Moreover, the cells displayed an increased expression of both AT1 and AT2 angiotensin II receptors and of p53 protein. The results suggest that angiotensin II could upregulate p53 expression in renal distal convoluted tubular cells and in the cells collecting ducts via both AT1 and AT2 receptors, which might be the crucial apoptosis-mediating mechanism in kidneys after excessive exercise. PMID: 16493576 [PubMed - indexed for MEDLINE

Exercise-induced apoptosis in rat kidney is mediated by both angiotensin II AT1 and AT2 receptors

Excessive physical exercise may lead to disturbance of the entire homeostasis in the body, including damage not only in skeletal muscles but also in many distant organs. The mechanisms responsible for the exercise-induced changes could include oxidative stress or angiotensin II. We previously showed that acute exercise led to apoptosis in kidney but not as a result of oxidative stress. In this study, we examined the role of angiotensin II and its AT1 and AT2 receptors in mediation of exercise-induced apoptosis in kidney. We clearly demonstrated that acute physical exercise induced apoptosis in renal cells of distal convoluted tubuli and cortical and medullary collecting ducts. Moreover, the cells displayed an increased expression of both AT1 and AT2 angiotensin II receptors and of p53 protein. The results suggest that angiotensin II could upregulate p53 expression in renal distal convoluted tubular cells and in the cells collecting ducts via both AT1 and AT2 receptors, which might be the crucial apoptosis-mediating mechanism in kidneys after excessive exercise

Analysis of the substrate-binding site of human carbonyl reductases CBR1 and CBR3 by site-directed mutagenesis.

Human carbonyl reductase is a member of the short-chain dehydrogenase/reductase (SDR) protein superfamily and is known to play an important role in the detoxification of xenobiotics bearing a carbonyl group. The two monomeric NADPH-dependent human isoforms of cytosolic carbonyl reductase CBR1 and CBR3 show a sequence similarity of 85% on the amino acid level, which is definitely high if compared to the low similarities usually observed among other members of the SDR superfamily (15-30%). Despite the sequence similarity and the similar features found in the available crystal structures of the two enzymes, CBR3 shows only low or no activity towards substrates that are metabolised by CBR1. This surprising substrate specificity is still not fully understood. In the present study, we introduced several point mutations and changed sequences of up to 17 amino acids of CBR3 to the corresponding amino acids of CBR1, to gather insight into the catalytic mechanism of both enzymes. Proteins were expressed in Escherichia coli and purified by Ni-affinity chromatography. Their catalytic properties were then compared using isatin and 9,10-phenanthrenequinone as model substrates. Towards isatin, wild-type CBR3 showed a catalytic efficiency of 0.018 microM(-1)min(-1), whereas wild-type CBR1 showed a catalytic efficiency of 13.5 microM(-1)min(-1). In particular, when nine residues (236-244) in the vicinity of the catalytic center and a proline (P230) in CBR3 were mutated to the corresponding residues of CBR1 a much higher k(cat)/K(m) value (5.7 microM(-1)min(-1)) towards isatin was observed. To gain further insight into the protein-ligand binding process, docking simulations were perfomed on this mutant and on both wild-type enzymes (CBR1 and CBR3). The theoretical model of the mutant was ad hoc built by means of standard comparative modelling