Roles of Putative Type II Secretion and Type IV Pilus Systems in the Virulence of Uropathogenic Escherichia coli

Type II secretion systems (T2SS) and the evolutionarily related type IV pili (T4P) are important virulence determinants in many Gram-negative bacterial pathogens. However, the roles of T2SS and T4P in the virulence of extraintestinal pathogenic Escherichia coli have not been determined.To investigate the functions of putative T2SS and T4P gene clusters present in the model uropathogenic E. coli (UPEC) strains UTI89 and CFT073, we deleted the secretin gene present in each cluster. The secretin forms a channel in the outer membrane that is essential for the function of T2S and T4P systems. We compared the secretin deletion mutants with their wild type counterparts using tissue culture assays and the CBA/J mouse model of ascending urinary tract infection. No deficiencies were observed with any of the mutants in adherence, invasion or replication in human bladder or kidney cell lines, but UTI89 DeltahofQ and UTI89 DeltagspD exhibited approximately 2-fold defects in fluxing out of bladder epithelial cells. In the mouse infection model, each of the knockout mutants was able to establish successful infections in the bladder and kidneys by day one post-infection. However, UTI89 DeltahofQ and a CFT073 DeltahofQ DeltayheF double mutant both exhibited defects in colonizing the kidneys by day seven post-infection.Based on our results, we propose that the putative T4P and T2S systems are virulence determinants of UPEC important for persistence in the urinary tract, particularly in renal tissues

Mutability and Importance of a Hypermutable Cell Subpopulation that Produces Stress-Induced Mutants in Escherichia coli

In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac+ mutants, with and without evidence of descent from the HMS, have similar Lac+ mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac+ mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones

Formation of an F′ Plasmid by Recombination between Imperfectly Repeated Chromosomal Rep Sequences: a Closer Look at an Old Friend (F′(128) pro lac)

Plasmid F′(128) was formed by an exchange between chromosomal Rep sequences that placed lac near dinB between many pairs of Rep sequences. Plasmid F′(128) is critical for selection-enhanced lac reversion (adaptive mutation), which requires prior lac amplification. The structure of F′(128) supports the idea that amplification is initiated by Rep-Rep recombination and that general mutagenesis requires coamplification of dinB (error-prone polymerase) with lac

Impact of the RNA Chaperone Hfq on the Fitness and Virulence Potential of Uropathogenic Escherichia coli▿ †

Hfq is a bacterial RNA chaperone involved in the posttranscriptional regulation of many stress-inducible genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Escherichia coli (UPEC) isolate UTI89 to effectively colonize the bladder and kidneys in a murine urinary tract infection model system. The disruption of hfq did not affect bacterial adherence to or invasion of host cells but did limit the development of intracellular microcolonies by UTI89 within the terminally differentiated epithelial cells that line the lumen of the bladder. In vitro, the hfq mutant was significantly impaired in its abilities to handle the antibacterial cationic peptide polymyxin B and reactive nitrogen and oxygen radicals and to grow in acidic medium (pH 5.0). Relative to the wild-type strain, the hfq mutant also had a substantially reduced migration rate on motility agar and was less prone to form biofilms. Hfq activities are known to impact the regulation of both the stationary-phase sigma factor RpoS (σS) and the envelope stress response sigma factor RpoE (σE). Although we saw similarities among hfq, rpoS, and rpoE deletion mutants in our assays, the rpoE and hfq mutants were phenotypically the most alike. Cumulatively, our data indicate that Hfq likely affects UPEC virulence-related phenotypes primarily by modulating membrane homeostasis and envelope stress response pathways

Mouse infection experiments with UTI89.

<p>Eight week-old, female CBA/J mice were infected by trans-urethral catheterization with the indicated WT or secretin knockout strains of UTI89. Bladders and kidneys were harvested on day 7 post-infection, weighed, homogenized and CFU/g of organ weight determined. The UTI89 Δ<i>hofQ</i> mutant was consistently defective in colonizing kidneys (p = 0.03) (B), whereas no differences were observed in CFU recovered from the bladder (A). The kidney colonization defect of UTI89 Δ<i>hofQ</i> (p = 0.027) was rescued by reintroduction of <i>hofQ</i> in trans (p = 0.013) (C). The bars indicate median CFU values. The limit of detection is indicated by the dotted line. In panels (A) and (B), the data from two independent experiments were combined together. Panel (C) shows data from one experiment. P values were calculated by Mann-Whitney test for non-parametric data with one-tailed P value.</p

Schematic Representation of the T2SS and T4P gene clusters in UTI89 and CFT073.

<p>The <i>E. coli</i> K12 and ETEC homologous T2SS gene clusters are shown in black and red, respectively. T4P genes are shown in blue. For the K12-T2SS, the <i>E. coli</i> K12 gene names are listed above the arrows and the generic <i>gsp</i> nomenclature is given below. The OM secretin genes are indicated by the hatched arrows. The open arrows are genes that do not show homology to any of the known T2SS or T4P genes.</p

Mouse infection experiments with CFT073.

<p>Eight week-old, female CBA/J mice were infected by trans-urethral catheterization with the indicated WT or secretin knockout strains of CFT073. Bladders and kidneys were harvested on day 7 post-infection, weighed, homogenized and CFU/g of organ weight determined. The CFT073 Δ<i>yheF</i> Δ<i>hofQ</i> double mutant was significantly defective in colonizing the kidneys (p = 0.013) (B), while bladder CFU (A) were lowered as compared to WT, but this difference was not significant. The kidney colonization defect of the CFT073 Δ<i>yheF</i> Δ<i>hofQ</i> double mutant (p = 0.005) was restored to WT-like levels by plasmid-borne <i>hofQ</i> (p = 0.038) (C). Addition of <i>yheF</i> in trans also increased colonization levels, but this was not statistically significant (p = 0.393) (C). The bars indicate median CFU values. The limit of detection is indicated by the dotted line. In panels (A) and (B), the data from two independent experiments were combined together. Panel (C) shows data from one experiment. P values were calculated by Mann-Whitney test for non-parametric data with one-tailed P value.</p