Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

The Cdkn2a gene product p19 alternative reading frame (p19ARF) is a critical regulator of IFNß-mediated Lyme arthritis.

Type I interferon (IFN) has been identified in patients with Lyme disease, and its abundant expression in joint tissues of C3H mice precedes development of Lyme arthritis. Forward genetics using C3H mice with severe Lyme arthritis and C57BL/6 (B6) mice with mild Lyme arthritis identified the Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1) on chromosome 4 (Chr4) as a regulator of B. burgdorferi-induced IFNß expression and Lyme arthritis severity. B6 mice introgressed with the C3H allele for Bbaa1 (B6.C3-Bbaa1 mice) displayed increased severity of arthritis, which is initiated by myeloid lineage cells in joints. Using advanced congenic lines, the physical size of the Bbaa1 interval has been reduced to 2 Mbp, allowing for identification of potential genetic regulators. Small interfering RNA (siRNA)-mediated silencing identified Cdkn2a as the gene responsible for Bbaa1 allele-regulated induction of IFNß and IFN-stimulated genes (ISGs) in bone marrow-derived macrophages (BMDMs). The Cdkn2a-encoded p19 alternative reading frame (p19ARF) protein regulates IFNß induction in BMDMs as shown by siRNA silencing and overexpression of ARF. In vivo studies demonstrated that p19ARF contributes to joint-specific induction of IFNß and arthritis severity in B. burgdorferi-infected mice. p19ARF regulates B. burgdorferi-induced IFNß in BMDMs by stabilizing the tumor suppressor p53 and sequestering the transcriptional repressor BCL6. Our findings link p19ARF regulation of p53 and BCL6 to the severity of IFNß-induced Lyme arthritis in vivo and indicate potential novel roles for p19ARF, p53, and BCL6 in Lyme disease and other IFN hyperproduction syndromes.

Ucl fimbriae regulation and glycan receptor specificity contribute to gut colonisation by extra-intestinal pathogenic Escherichia coli.

Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.

Bile Salts Regulate Zinc Uptake and Capsule Synthesis in a Mastitis-Associated Extraintestinal Pathogenic Escherichia coli Strain.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake (znuABC) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The ΔznuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated suppressor mutants with improved growth in bile salts, several of which no longer produced the K96 capsule made by strain M12. The addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with that of its unencapsulated ΔkpsCS mutant in two models of ExPEC disease. The wild-type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the ΔkpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC strains may be capable of causing human ExPEC illness. The virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

The Rhomboid Protease GlpG Promotes the Persistence of Extraintestinal Pathogenic Escherichia coli within the Gut.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are typically benign within the mammalian gut but can disperse to extraintestinal sites to cause diseases like urinary tract infections and sepsis. As occupation of the intestinal tract is often a prerequisite for ExPEC-mediated pathogenesis, we set out to understand how ExPEC colonizes this niche. A screen using transposon sequencing (Tn-seq) was performed to search for genes within ExPEC isolate F11 that are important for growth in intestinal mucus, which is thought to be a major source of nutrients for E. coli in the gut. Multiple genes that contribute to ExPEC fitness in mucus broth were identified, with genes that are directly or indirectly associated with fatty acid beta-oxidation pathways being especially important. One of the identified mucus-specific fitness genes encodes the rhomboid protease GlpG. In vitro, we found that the disruption of glpG had polar effects on the downstream gene glpR, which encodes a transcriptional repressor of factors that catalyze glycerol degradation. Mutation of either glpG or glpR impaired ExPEC growth in mucus and on plates containing the long-chain fatty acid oleate as the sole carbon source. In contrast, in a mouse gut colonization model in which the natural microbiota is unperturbed, the disruption of glpG but not glpR significantly reduced ExPEC survival. This work reveals a novel biological role for a rhomboid protease and highlights new avenues for defining mechanisms by which ExPEC strains colonize the mammalian gastrointestinal tract.

E. coli Common pili promote the fitness and virulence of a hybrid aEPEC/ExPEC strain within diverse host environments.

Pathogenic subsets of Escherichia coli include diarrheagenic (DEC) strains that cause disease within the gut and extraintestinal pathogenic E. coli (ExPEC) strains that are linked with urinary tract infections, bacteremia, and other infections outside of intestinal tract. Among DEC strains is an emergent pathotype known as atypical enteropathogenic E. coli (aEPEC), which can cause severe diarrhea. Recent sequencing efforts revealed that some E. coli strains possess genetic features that are characteristic of both DEC and ExPEC isolates. BA1250 is a newly reclassified hybrid strain with characteristics of aEPEC and ExPEC. This strain was isolated from a child with diarrhea, but its genetic features indicate that it might have the capacity to cause disease at extraintestinal sites. The spectrum of adhesins encoded by hybrid strains like BA1250 are expected to be especially important in facilitating colonization of diverse niches. E. coli common pilus (ECP) is an adhesin expressed by many E. coli pathogens, but how it impacts hybrid strains has not been ascertained. Here, using zebrafish larvae as surrogate hosts to model both gut colonization and extraintestinal infections, we found that ECP can act as a multi-niche colonization and virulence factor for BA1250. Furthermore, our results indicate that ECP-related changes in activation of envelope stress response pathways may alter the fitness of BA1250. Using an in silico approach, we also delineated the broader repertoire of adhesins that are encoded by BA1250, and provide evidence that the expression of at least a few of these varies in the absence of functional ECP.

Plant Phenolics Inhibit Focal Adhesion Kinase and Suppress Host Cell Invasion by Uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic and polyphenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here we tested a panel of four well-studied phenolic compounds - caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate - for effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses, and likely contribute to the development of chronic and recurrent infections. Using cell culture-based assays, we found that only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK, or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model, and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.

E. coli Common pili promote the fitness and virulence of a hybrid aEPEC/ExPEC strain within diverse host environments

Pathogenic subsets of Escherichia coli include diarrheagenic (DEC) strains that cause disease within the gut and extraintestinal pathogenic E. coli (ExPEC) strains that are linked with urinary tract infections, bacteremia, and other infections outside of intestinal tract. Among DEC strains is an emergent pathotype known as atypical enteropathogenic E. coli (aEPEC), which can cause severe diarrhea. Recent sequencing efforts revealed that some E. coli strains possess genetic features that are characteristic of both DEC and ExPEC isolates. BA1250 is a newly reclassified hybrid strain with characteristics of aEPEC and ExPEC. This strain was isolated from a child with diarrhea, but its genetic features indicate that it might have the capacity to cause disease at extraintestinal sites. The spectrum of adhesins encoded by hybrid strains like BA1250 are expected to be especially important in facilitating colonization of diverse niches. E. coli common pilus (ECP) is an adhesin expressed by many E. coli pathogens, but how it impacts hybrid strains has not been ascertained. Here, using zebrafish larvae as surrogate hosts to model both gut colonization and extraintestinal infections, we found that ECP can act as a multi-niche colonization and virulence factor for BA1250. Furthermore, our results indicate that ECP-related changes in activation of envelope stress response pathways may alter the fitness of BA1250. Using an in silico approach, we also delineated the broader repertoire of adhesins that are encoded by BA1250, and provide evidence that the expression of at least a few of these varies in the absence of functional ECP.

Invasion of Host Cells and Tissues by Uropathogenic Bacteria.

Within the mammalian urinary tract uropathogenic bacteria face many challenges, including the shearing flow of urine, numerous antibacterial molecules, the bactericidal effects of phagocytes, and a scarcity of nutrients. These problems may be circumvented in part by the ability of uropathogenic Escherichia coli and several other uropathogens to invade the epithelial cells that line the urinary tract. By entering host cells, uropathogens can gain access to additional nutrients and protection from both host defenses and antibiotic treatments. Translocation through host cells can facilitate bacterial dissemination within the urinary tract, while the establishment of stable intracellular bacterial populations may create reservoirs for relapsing and chronic urinary tract infections. Here we review the mechanisms and consequences of host cell invasion by uropathogenic bacteria, with consideration of the defenses that are brought to bear against facultative intracellular pathogens within the urinary tract. The relevance of host cell invasion to the pathogenesis of urinary tract infections in human patients is also assessed, along with some of the emerging treatment options that build upon our growing understanding of the infectious life cycle of uropathogenic E. coli and other uropathogens.