Bijaya K. Dhakal

Mechanisms and consequences of bladder cell invasion by uropathogenic Escherichia coli

Strains of uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections worldwide. Multiple studies over the past decade have called into question the dogmatic view that UPEC strains act as strictly extracellular pathogens. Rather, bacterial expression of filamentous adhesive organelles known as type 1 pili and Afa/Dr fibrils enable UPEC to invade host epithelial cells within the urinary tract. Entry into bladder epithelial cells provides UPEC with a protected niche where the bacteria can persist quiescently for long periods, unperturbed by host defences and protected from many antibiotic treatments. Alternately, internalized UPEC can rapidly multiply, forming large intracellular inclusions that can contain several thousand bacteria. Initial work aimed at defining the host and bacterial factors that modulate the entry, intracellular trafficking, and eventual resurgence of UPEC suggests a high degree of host‐pathogen crosstalk. Targeted disruption of these processes may provide a novel means to prevent and treat recurrent, relapsing and chronic infections within the urinary tract.

Inactivation of Host Akt/Protein Kinase B Signaling by Bacterial Pore-forming Toxins

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs), and they have the capacity to induce the death and exfoliation of target uroepithelial cells. This process can be facilitated by the pore-forming toxin alpha-hemolysin (HlyA), which is expressed and secreted by many UPEC isolates. Here, we demonstrate that HlyA can potently inhibit activation of Akt (protein kinase B), a key regulator of host cell survival, inflammatory responses, proliferation, and metabolism. HlyA ablates Akt activation via an extracellular calcium-dependent, potassium-independent process requiring HlyA insertion into the host plasma membrane and subsequent pore formation. Inhibitor studies indicate that Akt inactivation by HlyA involves aberrant stimulation of host protein phosphatases. We found that two other bacterial pore-forming toxins (aerolysin from Aeromonas species and alpha-toxin from Staphylococcus aureus) can also markedly attenuate Akt activation in a dose-dependent manner. These data suggest a novel mechanism by which sublytic concentrations of HlyA and other pore-forming toxins can modulate host cell survival and inflammatory pathways during the course of a bacterial infection.

Clathrin, AP‐2, and the NPXY‐binding subset of alternate endocytic adaptors facilitate FimH‐mediated bacterial invasion of host cells

The FimH adhesin, localized at the distal tips of type 1 pili, binds mannose‐containing glycoprotein receptors like α3β1 integrins and stimulates bacterial entry into target host cells. Strains of uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections, utilize FimH to invade bladder epithelial cells. Here we set out to define the mechanism by which UPEC enters host cells by investigating four of the major entry routes known to be exploited by invasive pathogens: caveolae, clathrin, macropinocytosis and secretory lysosomes. Using pharmacological inhibitors in combination with RNA interference against specific endocytic pathway components, mutant host cell lines and a mouse infection model system, we found that type 1 pili‐dependent bacterial invasion of host cells occurs via a cholesterol‐ and dynamin‐dependent phagocytosis‐like mechanism. This process did not require caveolae or secretory lysosomes, but was modulated by calcium levels, clathrin, and cooperative input from the primary clathrin adaptor AP‐2 and a subset of alternate adaptors comprised of Numb, ARH and Dab2. These alternate clathrin adaptors recognize NPXY motifs, as found within the cytosolic tail of β1 integrin, suggesting a functional link between the engagement of integrin receptors by FimH and the clathrin‐dependent uptake of type 1‐piliated bacteria.

Roles of putative type II secretion and type IV pilus systems in the virulence of uropathogenic Escherichia coli.

Background 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. Methodology/Principal Findings 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 ΔhofQ and UTI89 ΔgspD 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 ΔhofQ and a CFT073 ΔhofQ ΔyheF double mutant both exhibited defects in colonizing the kidneys by day seven post-infection. Conclusions/Significance 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.

Uropathogenic Escherichia coli Invades Host Cells via an HDAC6-modulated Microtubule-dependent Pathway *□

Strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili that promote bacterial colonization and invasion of the bladder epithelium. Type 1 pilus-mediated interactions with host receptors, including α3β1 integrin, trigger localized actin rearrangements that lead to internalization of adherent bacteria via a zipper-like mechanism. Here we report that type 1 pilus-mediated bacterial invasion of bladder cells also requires input from host microtubules and histone deacetylase 6 (HDAC6), a cytosolic enzyme that, by deacetylating α-tubulin, can alter the stability of microtubules along with the recruitment and directional trafficking of the kinesin-1 motor complex. We found that disruption of microtubules by nocodazole or vinblastine treatment, as well as microtubule stabilization by taxol, inhibited host cell invasion by UPEC, as did silencing of HDAC6 expression or pharmacological inhibition of HDAC6 activity. Invasion did not require two alternate HDAC6 substrates, Hsp90 and cortactin, but was dependent upon the kinesin-1 light chain KLC2 and an upstream activator of HDAC6, aurora A kinase. These results indicate that HDAC6 and microtubules act as vital regulatory elements during the invasion process, possibly via indirect effects on kinesin-1 and associated cargos.

The Cpx Stress Response System Potentiates the Fitness and Virulence of Uropathogenic Escherichia coli

ABSTRACT Strains of uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections, representing one of the most widespread and successful groups of pathogens on the planet. To colonize and persist within the urinary tract, UPEC must be able to sense and respond appropriately to environmental stresses, many of which can compromise the bacterial envelope. The Cpx two-component envelope stress response system is comprised of the inner membrane histidine kinase CpxA, the cytosolic response regulator CpxR, and the periplasmic auxiliary factor CpxP. Here, by using deletion mutants along with mouse and zebrafish infection models, we show that the Cpx system is critical to the fitness and virulence of two reference UPEC strains, the cystitis isolate UTI89 and the urosepsis isolate CFT073. Specifically, deletion of the cpxRA operon impaired the ability of UTI89 to colonize the murine bladder and greatly reduced the virulence of CFT073 during both systemic and localized infections within zebrafish embryos. These defects coincided with diminished host cell invasion by UTI89 and increased sensitivity of both strains to complement-mediated killing and the aminoglycoside antibiotic amikacin. Results obtained with the cpxP deletion mutants were more complicated, indicating variable strain-dependent and niche-specific requirements for this well-conserved auxiliary factor.

Bacteria differentially induce degradation of Bcl-xL, a survival protein, by human platelets.

Bacteria can enter the bloodstream in response to infectious insults. Bacteremia elicits several immune and clinical complications, including thrombocytopenia. A primary cause of thrombocytopenia is shortened survival of platelets. We demonstrate that pathogenic bacteria induce apoptotic events in platelets that include calpain-mediated degradation of Bcl-x(L), an essential regulator of platelet survival. Specifically, bloodstream bacterial isolates from patients with sepsis induce lateral condensation of actin, impair mitochondrial membrane potential, and degrade Bcl-x(L) protein in platelets. Bcl-x(L) protein degradation is enhanced when platelets are exposed to pathogenic Escherichia coli that produce the pore-forming toxin α-hemolysin, a response that is markedly attenuated when the gene is deleted from E coli. We also found that nonpathogenic E coli gain degrading activity when they are forced to express α-hemolysin. Like α-hemolysin, purified α-toxin readily degrades Bcl-x(L) protein in platelets, as do clinical Staphylococcus aureus isolates that produce α-toxin. Inhibition of calpain activity, but not the proteasome, rescues Bcl-x(L) protein degradation in platelets coincubated with pathogenic E coli including α-hemolysin producing strains. This is the first evidence that pathogenic bacteria can trigger activation of the platelet intrinsic apoptosis program and our results suggest a new mechanism by which bacterial pathogens might cause thrombocytopenia in patients with bloodstream infections.

First Case Reports of Ignatzschineria (Schineria) indica Associated with Myiasis

ABSTRACT We report three cases of infection due to the Gram-negative rod Ignatzschineria (Schineria) indica involving bacteremia and the urinary tract. Two cases were clearly associated with maggot infestation, and the third could conceivably have had unrecognized maggot infestation of the urinary tract. We believe these cases to be the first I. indica infections reported in association with maggot infestation and myiasis.

Histone Deacetylase 6 Regulates Bladder Architecture and Host Susceptibility to Uropathogenic Escherichia coli.

Histone deacetylase 6 (HDAC6) is a non-canonical, mostly cytosolic histone deacetylase that has a variety of interacting partners and substrates. Previous work using cell-culture based assays coupled with pharmacological inhibitors and gene-silencing approaches indicated that HDAC6 promotes the actin- and microtubule-dependent invasion of host cells by uropathogenic Escherichia coli (UPEC). These facultative intracellular pathogens are the major cause of urinary tract infections. Here, we examined the involvement of HDAC6 in bladder colonization by UPEC using HDAC6 knockout mice. Though UPEC was unable to invade HDAC6−/− cells in culture, the bacteria had an enhanced ability to colonize the bladders of mice that lacked HDAC6. This effect was transient, and by six hours post-inoculation bacterial titers in the HDAC6−/− mice were reduced to levels seen in wild type control animals. Subsequent analyses revealed that the mutant mice had greater bladder volume capacity and fluid retention, along with much higher levels of acetylated α-tubulin. In addition, infiltrating neutrophils recovered from the HDAC6−/− bladder harbored significantly more viable bacteria than their wild type counterparts. Cumulatively, these changes may negate any inhibitory effects that the lack of HDAC6 has on UPEC entry into individual host cells, and suggest roles for HDAC6 in other urological disorders such as urinary retention.