Jai J. Tree

Identification of Bacteriophage-Encoded Anti-sRNAs in Pathogenic Escherichia coli

Summary In bacteria, Hfq is a core RNA chaperone that catalyzes the interaction of mRNAs with regulatory small RNAs (sRNAs). To determine in vivo RNA sequence requirements for Hfq interactions, and to study riboregulation in a bacterial pathogen, Hfq was UV crosslinked to RNAs in enterohemorrhagic Escherichia coli (EHEC). Hfq bound repeated trinucleotide motifs of A-R-N (A-A/G-any nucleotide) often associated with the Shine-Dalgarno translation initiation sequence in mRNAs. These motifs overlapped or were adjacent to the mRNA sequences bound by sRNAs. In consequence, sRNA-mRNA duplex formation will displace Hfq, promoting recycling. Fifty-five sRNAs were identified within bacteriophage-derived regions of the EHEC genome, including some of the most abundant Hfq-interacting sRNAs. One of these (AgvB) antagonized the function of the core genome regulatory sRNA, GcvB, by mimicking its mRNA substrate sequence. This bacteriophage-encoded “anti-sRNA” provided EHEC with a growth advantage specifically in bovine rectal mucus recovered from its primary colonization site in cattle.

An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157: H7 with bovine intestinal epithelium

Enterohaemorrhagic Escherichia coli O157 : H7 is a bacterial pathogen that can cause haemorrhagic colitis and haemolytic uremic syndrome. In the primary reservoir host, cattle, the terminal rectum is the principal site of E. coli O157 colonization. In this study, bovine terminal rectal primary epithelial cells were used to examine the role of H7 flagella in epithelial adherence. Binding of a fliCH7 mutant O157 strain to rectal epithelium was significantly reduced as was binding of the flagellated wild‐type strain following incubation with H7‐specific antibodies. Complementation of fliCH7 mutant O157 strain with fliCH7 restored the adherence to wild‐type levels; however, complementation with fliCH6 did not restore it. High‐resolution ultrastructural and imunofluorescence studies demonstrated the presence of abundant flagella forming physical contact points with the rectal epithelium. Binding to terminal rectal epithelium was specific to H7 by comparison with other flagellin types tested. In‐cell Western assays confirmed temporal expression of flagella during O157 interaction with epithelium, early expression was suppressed during the later stages of microcolony and attaching and effacing lesion formation. H7 flagella are expressed in vivo by individual bacteria in contact with rectal mucosa. Our data demonstrate that the H7 flagellum acts as an adhesin to bovine intestinal epithelium and its involvement in this crucial initiating step for colonization indicates that H7 flagella could be an important target in intervention strategies.

Virulence properties of asymptomatic bacteriuria Escherichia coli.

In asymptomatic bacteriuria (ABU), bacteria colonize the urinary tract without provoking symptoms. Here, we compared the virulence properties of a collection of ABU Escherichia coli strains to cystitis and pyelonephritis strains. Specific urinary tract infection (UTI)-associated virulence genes, hemagglutination characteristics, siderophore production, hemolysis, biofilm formation, and the ability of strains to adhere to and induce cytokine responses in epithelial cells were analyzed. ABU strains were phylogenetically related to strains that cause symptomatic UTI. However, the virulence properties of the ABU strains were variable and dependent on a combination of genotypic and phenotypic factors. Most ABU strains adhered poorly to epithelial cells; however, we also identified a subgroup of strongly adherent strains that were unable to stimulate an epithelial cell IL-6 cytokine response. Poor immune activation may represent one mechanism whereby ABU E. coli evade immune detection after the establishment of bacteriuria.

The multi-copper-ion oxidase CueO of Salmonella enterica serovar Typhimurium is required for systemic virulence

ABSTRACT Salmonella enterica serovar Typhimurium possesses a multi-copper-ion oxidase (multicopper oxidase), CueO (also known as CuiD), a periplasmic enzyme known to be required for resistance to copper ions. CueO from S. Typhimurium was expressed as a recombinant protein in Escherichia coli, and the purified protein exhibited a high cuprous oxidase activity. We have characterized an S. Typhimurium cueO mutant and confirmed that it is more sensitive to copper ions. Using a murine model of infection, it was observed that the cueO mutant was significantly attenuated, as indicated by reduced recovery of bacteria from liver and spleen, although there was no significant difference in recovery from Peyers patches and mesenteric lymph nodes. However, the intracellular survival of the cueO mutant in unprimed or gamma-interferon-primed murine macrophages was not statistically different from that of wild-type Salmonella, suggesting that additional host factors are involved in clearance of the cueO mutant. Unlike a cueO mutant from E. coli, the S. Typhimurium cueO mutant did not show greater sensitivity to hydrogen peroxide and its sensitivity to copper ions was not affected by siderophores. Similarly, the S. Typhimurium cueO mutant was not rescued from copper ion toxicity by addition of the branched-chain amino acids and leucine.

UpaH Is a Newly Identified Autotransporter Protein That Contributes to Biofilm Formation and Bladder Colonization by Uropathogenic Escherichia coli CFT073

ABSTRACT Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.

Identification of bacterial target proteins for the salicylidene acylhydrazide class of virulence blocking compounds

A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.

Controlling injection: regulation of type III secretion in enterohaemorrhagic Escherichia coli

Type III secretion (T3S) systems enable the injection of bacterial proteins through membrane barriers into host cells, either from outside the host cell or from within a vacuole. This system is required for colonization of their ruminant reservoir hosts by enterohaemorrhagic Escherichia coli (EHEC) and might also be important for the etiology of disease in the incidental human host. T3S systems of E. coli inject a cocktail of proteins into epithelial cells that enables bacterial attachment and promotes longer-term colonization in the animal. Here, we review recent progress in our understanding of the regulation of T3S in EHEC, focusing on the induction and assembly of the T3S system, the co-ordination of effector protein expression, and the timing of effector protein export through the apparatus. Strain variation is often associated with differences in bacteriophages encoding the production of Shiga toxin and in multiple cryptic prophage elements that can encode effector proteins and T3S regulators. It is evident that this repertoire of phage-related sequences results in the different levels of T3S demonstrated between strains, with implications for EHEC epidemiology and strain evolution.

Characterization of the Effects of Salicylidene Acylhydrazide Compounds on Type III Secretion in Escherichia coli O157:H7

ABSTRACT Recent work has highlighted a number of compounds that target bacterial virulence by affecting gene regulation. In this work, we show that small-molecule inhibitors affect the expression of the type III secretion system (T3SS) of Escherichia coli O157:H7 in liquid culture and when this bacterium is attached to bovine epithelial cells. Inhibition of T3SS expression resulted in a reduction in the capacity of the bacteria to form attaching and effacing lesions. Our results show that there is marked variation in the abilities of four structurally related compounds to inhibit the T3SS of a panel of isolates. Using transcriptomics, we performed a comprehensive analysis of the conserved and inhibitor-specific transcriptional responses to these four compounds. These analyses of gene expression show that numerous virulence genes, located on horizontally acquired DNA elements, are affected by the compounds, but the number of genes significantly affected varied markedly for the different compounds. Overall, we highlight the importance of assessing the effect of such “antivirulence” agents on a range of isolates and discuss the possible mechanisms which may lead to the coordinate downregulation of horizontally acquired virulence genes.

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

RNA sequencing studies have identified hundreds of non‐coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA–RNA interactions in bacteria. Here we demonstrate that in vivo sRNA–mRNA duplexes can be recovered using UV‐crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA–mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA–mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNAs. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli. Numerous sRNA interactions were also identified with non‐coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.

Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

ABSTRACT Staphylococcus aureus is a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates of S. aureus. Here, we describe the construction and characterization of the IMXXB series of Escherichia coli strains that mimic the type I adenine methylation profiles of S. aureus clonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of major S. aureus lineages. IMPORTANCE The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus. The IMXXB series of E. coli strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus. The genetic manipulation of clinical S. aureus isolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset of S. aureus strains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite of E. coli strains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer into S. aureus. The IMXXB series of E. coli strains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation of S. aureus.