Cellular RNA Targets of Cold Shock Proteins CspC and CspE and Their Importance for Serum Resistance in Septicemic Escherichia coli.

The RNA chaperones, cold shock proteins CspC and CspE, are important in stress response and adaptation. We studied their role in the pathogenesis of a virulent Escherichia coli, representative of extraintestinal pathogenic E. coli (ExPEC) which are serum resistant and septicemic. We performed a global analysis to identify transcripts that interact with these cold shock proteins (CSPs), focusing on virulence-related genes. We used CLIP-seq, which combines UV cross-linking, immunoprecipitation and RNA sequencing. A large number of transcripts bound to the CSPs were identified, and many bind both CspC and CspE. Many transcripts were of genes involved in protein synthesis, transcription and energy metabolism. In addition, there were virulence-related genes, (i.e., fur and ryhB), essential for iron homeostasis. The CLIP-seq results were validated on two transcripts, clpX and tdcA, reported as virulence-associated. Deletion of either CspC or CspE significantly decreased their transcript levels and in a double deletion mutant cspC/cspE, the transcript stability of tdcA and clpX was reduced by 32-fold and 10-fold, respectively. We showed that these two genes are important for virulence, as deleting either of them resulted in loss of serum resistance, a requirement for sepsis. As several virulence-related transcripts interact with CspC or CspE, we determined the importance of these proteins for growth in serum and showed that deletion of either gene significantly reduced serum survival. This phenotype could be partially complemented by cspE and fully complemented by cspC. These results indicate that the two RNA chaperones are essential for virulence, and that CspC particularly critical. IMPORTANCE Virulent Escherichia coli strains that cause infections outside the intestinal tract-extraintestinal pathogenic E. coli (ExPEC)-constitute a major clinical problem worldwide. They are involved in several distinct conditions, including urinary tract infections, newborn meningitis, and sepsis. Due to increasing antibiotic resistance, these strains are a main factor in hospital and community-acquired infections. Because many strains, which do not cross-react immunologically are involved, developing a simple vaccine is not possible. Therefore, it is essential to understand the pathogenesis of these bacteria to identify potential targets for developing drugs or vaccines. One of the least investigated systems involves RNA binding proteins, important for stability of transcripts and global gene regulation. Two such proteins are CspC and CspE ("cold shock proteins"), RNA chaperones involved in stress adaptation. Here we performed a global analysis to identify the transcripts which are affected by these two chaperones, with focus on virulence-associated transcripts.

Surviving Serum: the Escherichia coli iss Gene of Extraintestinal Pathogenic E. coli Is Required for the Synthesis of Group 4 Capsule.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains constitute a serious and emerging clinical problem, as they cause a variety of infections and are usually highly antibiotic resistant. Many ExPEC strains are capable of evading the bactericidal effects of serum and causing sepsis. One critical factor for the development of septicemia is the increased serum survival (iss) gene, which is highly correlated with complement resistance and lethality. Although it is very important, the function of the iss gene has not been elucidated so far. We have been studying the serum survival of a septicemic strain of E. coli serotype O78, which has a group 4 capsule. Here, we show that the iss gene is required for the synthesis of capsules, which protect the bacteria from the bactericidal effect of complement. Moreover, we show that the deletion of the iss gene results in significantly increased binding of the complement proteins that constitute the membrane attack complex to the bacterial surface.

Genomics-based epidemiology of bovine Mycoplasma bovis strains in Israel.

BACKGROUND: Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis affecting cattle production and animal welfare. In the past in Israel, M. bovis has been most frequently associated with bovine respiratory disease (BRD) and was rarely isolated from mastitis. This situation changed in 2008 when M. bovis-associated mastitis emerged in Israel. The aim of this study was to utilize whole genome sequencing to evaluate the molecular epidemiology and genomic diversity of M. bovis mastitis-associated strains and their genetic relatedness to M. bovis strains isolated from BRD in local feedlot calves and those imported to Israel from different European countries and Australia. RESULTS: Phylogeny based on total single nucleotide polymorphism (SNP) analysis of 225 M. bovis genomes clearly showed clustering of isolates on the basis of geographical origin: strains isolated from European countries clustered together and separately from Australian and Chinese isolates, while Israeli isolates were found in the both groups. The dominant genotype was identified among local mastitis-associated M. bovis isolates. This genotype showed a close genomic relatedness to M. bovis strains isolated from calves imported to Israel from Australia, to original Australian M. bovis strains, as well as to strains isolated in China. CONCLUSIONS: This study represents the first comprehensive high-resolution genome-based epidemiological analysis of M. bovis in Israel and illustrates the possible dissemination of the pathogen across the globe by cattle trade.

Repeat modularity as a beneficial property of multiple CRISPR-Cas systems.

CRISPR-Cas systems are a highly effective immune mechanism for prokaryotes, providing defense against invading foreign DNA. By definition, all CRISPR-Cas systems have short repeats interspersing their spacers. These repeats play a key role in preventing cleavage of self DNA and in the integration of new spacers. Here we focus on the phenomenon of repeat modularity, namely the unexpectedly high degree of repeat conservation across different systems within a genome or between different species. We hypothesize that modularity can be beneficial for CRISPR-Cas containing organisms, because it facilitates horizontal acquisition of 'pre-immunized' CRISPR arrays and allows the utilization of spacers acquired by one system for use by other systems within the same cell.

Pandemic Bacteremic Escherichia Coli Strains: Evolution and Emergence of Drug-Resistant Pathogens.

In recent years, there have been several pandemics of E. coli strains which are highly virulent and antibiotics resistant. Here, we discuss one recent pandemic strain, ST131. These E. coli strains are members of the virulence-associated phylogenetic group B2 and exhibit extraintestinal virulence factors, including various adhesins, toxins, siderophores, and protectins. This group often also harbors a diverse range of antimicrobial resistance types and mechanisms and may have particular metabolic capacities that enable it to colonize many individuals asymptomatically, while out competing other E. coli strains. Here, we discuss this clonal group in the context of other pathogenic E. coli and focus on its specific characteristics in terms of resistance, virulence, and metabolism.

The Escherichia coli Type III Secretion System 2 Has a Global Effect on Cell Surface.

Many strains of Escherichia coli carry a 29,250-bp ETT2 pathogenicity island (PAI), which includes genes predicted to encode type III secretion system (T3SS) components. Because it is similar to the Salmonella pathogenicity island 1 (SPI-1) system, encoding a T3SS in Salmonella enterica, it was assumed that ETT2 also encodes a secretion system injecting effectors into host cells. This assumption was checked in E. coli serotype O2-associated with urinary tract infections and septicemia-which has an intact ETT2 gene cluster, in contrast to most strains in which this cluster carries deletions and mutations. A proteomic search did not reveal any putative secreted effector. Instead, the majority of the secreted proteins were identified as flagellar proteins. A deletion of the ETT2 gene cluster significantly reduced the secretion of flagellar proteins, resulting in reduced motility. There was also a significant reduction in the transcriptional level of flagellar genes, indicating that ETT2 affects the synthesis, rather than secretion, of flagellar proteins. The ETT2 deletion also resulted in additional major changes in secretion of fimbrial proteins and cell surface proteins, resulting in relative resistance to detergents and hydrophobic antibiotics (novobiocin), secretion of large amounts of outer membrane vesicles (OMVs), and altered multicellular behavior. Most important, the ETT2 deletion mutants were sensitive to serum. These major changes indicate that the ETT2 gene cluster has a global effect on cell surface and physiology, which is especially important for pathogenicity, as it contributes to the ability of the bacteria to survive serum and cause sepsis.IMPORTANCE Drug-resistant extraintestinal pathogenic E. coli (ExPEC) strains are major pathogens, especially in hospital- and community-acquired infections. They are the major cause of urinary tract infections and are often involved in septicemia with high mortality. ExPEC strains are characterized by broad-spectrum antibiotic resistance, and development of a vaccine is not trivial because the ExPEC strains include a large number of serotypes. It is therefore important to understand the virulence factors that are involved in pathogenicity of ExPEC and identify new targets for development of antibacterial drugs or vaccines. Such a target could be ETT2, a unique type III secretion system present (complete or in parts) in many ExPEC strains. Here, we show that this system has a major effect on the bacterial surface-it affects sensitivity to drugs, motility, and secretion of extracellular proteins and outer membrane vesicles. Most importantly, this system is important for serum resistance, a prerequisite for septicemia.

Escherichia coli isolates from patients with bacteremic urinary tract infection are genetically distinct from those derived from sepsis following prostate transrectal biopsy.

BACKGROUND: Transrectal ultrasound-guided (TRUS) prostate biopsy is a very common procedure that is generally considered relatively safe. However, severe sepsis can occur after TRUS prostate biopsies, with Escherichia coli being the predominant causative agent. A common perception is that the bacteria that cause post-TRUS prostate biopsy infections originate in the urinary tract, but this view has not been adequately tested. Yet other authors believe on the basis of indirect evidence that the pathogens are introduced into the bloodstream by the biopsy needle after passage through the rectal mucosa. METHODS: We compared E. coli isolates from male patients with bacteremic urinary tract infection (B-UTI) to isolates of patients with post prostate biopsy sepsis (PPBS), in terms of their sequence types, determined by multi-locus sequence typing (MLST) and their virulence markers. RESULTS: B-UTI isolates were much richer in virulence genes than were PPBS isolates, supporting the hypothesis that E. coli causing PPBS derive directly from the rectum. Sequence type 131 (ST131) strains and related strain from the ST131 were common (>30%) among the E. coli isolates from PPBS patients as well as from B-UTI patients and all these strains expressed extended spectrum beta-lactamases. CONCLUSIONS: Our finding supports the hypothesis that E. coli causing PPBS derive directly from the rectum, bypassing the urinary tract, and therefore do not require many of the virulence capabilities necessary for an E. coli strain that must persist in the urinary tract. In light of the increasing prevalence of highly resistant E. coli strains, a new approach for prevention of PPBS is urgently required.

A halocin-H4 mutant Haloferax mediterranei strain retains the ability to inhibit growth of other halophilic archaea.

Many members of the Halobacteriaceae were found to produce halocins, molecules that inhibit the growth of other halophilic archaea. Halocin H4 that is produced by Haloferax mediterranei and inhibits the growth of Halobacterium salinarum is one of the best studied halocins to date. The gene encoding this halocin had been previously identified as halH4, located on one of Hfx. mediterranei megaplasmids. We generated a mutant of the halH4 gene and examined the killing ability of the Haloferax mediterranei halH4 mutant with respect to both Halobacterium salinarum and Haloferax volcanii. We showed that both wild-type Hfx. mediterranei and the halH4 mutant strain efficiently inhibited the growth of both species, indicating halocin redundancy. Surprisingly, the halH4 deletion mutant exhibited faster growth in standard medium than the wild type, and is likely to have a better response to several nucleotides, which could explain this phenotype.