Distinguishing the contribution of type 1 pili from that of other QseB-misregulated factors when QseC is absent during urinary tract infection

Urinary tract infections (UTI), primarily caused by uropathogenic Escherichia coli (UPEC), are one of the leading bacterial infections due to their high frequency and rate of recurrence. Both type 1 pilus adhesive organelles (fim) and the QseC sensor kinase have been implicated in UPEC virulence during UTI and have been individually reported to be promising drug targets. Deletion of qseC leads to pleiotropic effects due to unregulated activation of the cognate response regulator QseB, influencing conserved metabolic processes and diminishing expression of virulence genes, including type 1 pili. Here, we discern the type 1 pilus-dependent and -independent effects that contribute to the virulence attenuation of a UPEC qseC deletion mutant in a murine model of experimental UTI. We show that although a ΔqseC mutant restored for type 1 pilus expression regains the ability to colonize the host and initiate acute infection up to 16 h postinfection, it is rapidly outcompeted during acute infection when coinoculated with a wild-type strain. As a result, this strain has a diminished capacity to establish chronic infection. A prophylactic oral dose of a FimH small-molecular-weight antagonist (ZFH-02056) further reduced the ability of the qseC mutant to establish chronic infection. Thus, loss of QseC significantly enhances the efficacy of ZFH-02056. Collectively, our work indicates that type 1 pili and QseC become critical in different infection stages, and that dual targeting of these factors has an additive effect on ablating UPEC virulence

Transposon mutagenesis identifies uropathogenic Escherichia coli biofilm factors

Uropathogenic Escherichia coli (UPEC), which accounts for 85% of urinary tract infections (UTI), assembles biofilms in diverse environments, including the host. Besides forming biofilms on biotic surfaces and catheters, UPEC has evolved an intracellular pathogenic cascade that culminates in the formation of biofilm-like intracellular bacterial communities (IBCs) within bladder epithelial cells. Rapid bacterial replication during IBC formation augments a build-up in bacterial numbers and persistence within the host. Relatively little is known about factors mediating UPEC biofilm formation and how these overlap with IBC formation. To address this gap, we screened a UPEC transposon mutant library in three in vitro biofilm conditions: Luria broth (LB)-polyvinyl chloride (PVC), YESCA (yeast extract-Casamino Acids)-PVC, and YESCA-pellicle that are dependent on type 1 pili (LB) and curli (YESCA), respectively. Flagella are important in all three conditions. Mutants were identified that had biofilm defects in all three conditions but had no significant effects on the expression of type 1 pili, curli, or flagella. Thus, this approach uncovered a comprehensive inventory of novel effectors and regulators that are involved in UPEC biofilm formation under multiple conditions. A subset of these mutants was found to be dramatically attenuated and unable to form IBCs in a murine model of UTI. Collectively, this study expands our insights into UPEC multicellular behavior that may provide insights into IBC formation and virulence

Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs - A review

Pseudomonas aeruginosa is the most prevalent pathogen of cystic fibrosis (CF) lung disease. Its long persistence in CF airways is associated with sophisticated mechanisms of adaptation, including biofilm formation, resistance to antibiotics, hypermutability and customized pathogenicity in which virulence factors are expressed according the infection stage. CF adaptation is triggered by high selective pressure of inflamed CF lungs and by antibiotic treatments. Bacteria undergo genetic, phenotypic, and physiological variations that are fastened by the repeating interplay of mutation and selection. During CF infection development, P. aeruginosa gradually shifts from an acute virulent pathogen of early infection to a host-adapted pathogen of chronic infection. This paper reviews the most common changes undergone by P. aeruginosa at each stage of infection development in CF lungs. The comprehensive understanding of the adaptation process of P. aeruginosa may help to design more effective antimicrobial treatments and to identify new targets for future drugs to prevent the progression of infection to chronic stages.The authors thank the project FCT PTDC/SAUSAP/113196/2009/FCOMP-01-0124-FEDER-016012, the Strategic Project PEst-OE/EQB/LA0023/2013, the Project. BioHealth-Biotechnology and Bioengineering approaches to improve health quality., Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER, the project. RECI/BBB-EBI/0179/2012-Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB., Ref. FCOMP-01-0124-FEDER-027462, FEDER. The authors also acknowledge PhD Grant of Ana MargaridaSousa SFRH/BD/72551/2010

Functional activities of the Tsh protein from avian pathogenic Escherichia coli (APEC) strains

The temperature-sensitive hemagglutinin (Tsh) expressed by strains of avian pathogenic Escherichia (E.) coli (APEC) has both agglutinin and protease activities. Tsh is synthesized as a 140 kDa precursor protein, whose processing results in a 106 kDa passenger domain (Tshs) and a 33 kDa β-domain (Tshβ). In this study, both recombinant Tsh (rTsh) and supernatants from APEC, which contain Tshs (106 kDa), caused proteolysis of chicken tracheal mucin. Both rTsh (140 kDa) and pellets from wild-type APEC, which contain Tshβ (33 kDa), agglutinated chicken erythrocytes. On Western blots, the anti-rTsh antibody recognized the rTsh and 106 kDa proteins in recombinant E. coli BL21/pET 101-Tsh and in the supernatants from APEC grown at either 37℃ or 42℃. Anti-rTsh also recognized a 33 kDa protein in the pellets from APEC13 cultures grown in either Luria-Bertani agar, colonization factor antigen agar, or mucin agar at either 26℃, 37℃, or 42℃, and in the extracts of outer membrane proteins of APEC. The 106 kDa protein was more evident when the bacteria were grown at 37℃ in mucin agar, and it was not detected when the bacteria were grown at 26℃ in any of the culture media used in this study. Chicken anti-Tsh serum inhibited hemagglutinating and mucinolytic activities of strain APEC13 and recombinant E. coli BL21/pET101-Tsh. This work suggests that the mucinolytic activity of Tsh might be important for the colonization of the avian tracheal mucous environment by APEC

KinD Is a Checkpoint Protein Linking Spore Formation to Extracellular-Matrix Production in Bacillus subtilis Biofilms

Bacillus subtilis cells form multicellular biofilm communities in which spatiotemporal regulation of gene expression occurs, leading to differentiation of multiple coexisting cell types. These cell types include matrix-producing and sporulating cells. Extracellular matrix production and sporulation are linked in that a mutant unable to produce matrix is delayed for sporulation. Here, we show that the delay in sporulation is not due to a growth advantage of the matrix-deficient mutant under these conditions. Instead, we show that the link between matrix production and sporulation is through the Spo0A signaling pathway. Both processes are regulated by the phosphorylated form of the master transcriptional regulator Spo0A. When cells have low levels of phosphorylated Spo0A (Spo0A~P), matrix genes are expressed; however, at higher levels of Spo0A~P, sporulation commences. We have found that Spo0A~P levels are maintained at low levels in the matrix-deficient mutant, thereby delaying expression of sporulation-specific genes. This is due to the activity of one of the components of the Spo0A phosphotransfer network, KinD. A deletion of kinD suppresses the sporulation defect of matrix mutants, while its overproduction delays sporulation. Our data indicate that KinD displays a dual role as a phosphatase or a kinase and that its activity is linked to the presence of extracellular matrix in the biofilms. We propose a novel role for KinD in biofilms as a checkpoint protein that regulates the onset of sporulation by inhibiting the activity of Spo0A until matrix, or a component therein, is sensed

Identification and Functional Characterization of Gene Components of Type VI Secretion System in Bacterial Genomes

A new secretion system, called the Type VI Secretion system (T6SS), was recently reported in Vibrio cholerae, Pseudomonas aeruginosa and Burkholderia mallei. A total of 18 genes have been identified to be belonging to this secretion system in V. cholerae. Here we attempt to identify presence of T6SS in other bacterial genomes. This includes identification of orthologous sequences, conserved motifs, domains, families, 3D folds, genomic islands containing T6SS components, phylogenetic profiles and protein-protein association of these components. Our analysis indicates presence of T6SS in 42 bacteria and its absence in most of their non-pathogenic species, suggesting the role of T6SS in imparting pathogenicity to an organism. Analysis of genomic regions containing T6SS components, phylogenetic profiles and protein-protein association of T6SS components indicate few additional genes which could be involved in this secretion system. Based on our studies, functional annotations were assigned to most of the components. Except one of the genes, we could group all the other genes of T6SS into those belonging to the puncturing device, and those located in the outer membrane, transmembrane and inner membrane. Based on our analysis, we have proposed a model of T6SS and have compared the same with the other bacterial secretion systems

Serine Protease Autotransporters of Enterobacteriaceae (SPATEs): Biogenesis and Function

Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins

A 3D individual-based model to investigate the spatially heterogeneous response of bacterial biofilms to antimicrobial agents

The response of bacterial biofilms to treatment with antimicrobial agents is often characterized by the emergence of recalcitrant cellular microcolonies. We present an individual-based model to investigate the biophysical mechanisms of the selective resistance that arises within the biofilm and leads to a spatially heterogeneous response upon treatment with antibiotics. The response occurs in 3 distinct phases. In the first phase, the subpopulation of metabolically active cells diminishes due to antibiotic-induced cell death. Subsequently, in the second phase, increased nutrient availability allows dormant cells in the lower layers of the biofilm to transform into metabolically active cells. In the third phase, survival of the biofilm is governed by the interplay between 2 contrasting factors: (1) rate of antibiotic-induced cell death and (2) rate of transformation of dormant cells into active ones. Metabolically active cells at the distal edge of the biofilm sacrifice themselves to protect the dormant cells in the interior by (1) reducing local antibiotic concentrations and (2) increasing nutrient availability. In the presence of quorum sensing, biofilms exhibit increased tolerance compared with the quorum sensing-negative strains. Extracellular polymeric substance (EPS) forms a protective layer at the top of the biofilm, thereby limiting antibiotic penetration. The surviving cells, in turn, produce EPS resulting in a feedback-like mechanism of resistance. Whereas resistance in QS- biofilms occurs because of transformation of dormant cells into metabolically active cells, this transformation is less pronounced in QS+ biofilms, and resistance is a consequence of the sequestration of the antibiotic by EPS