Dual adhesive unipolar polysaccharides synthesized by overlapping biosynthetic pathways in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a member of the Alphaproteobacteria that pathogenises plants and associates with biotic and abiotic surfaces via a single cellular pole. A. tumefaciens produces the unipolar polysaccharide (UPP) at the site of surface contact. UPP production is normally surface-contact inducible, but elevated levels of the second messenger cyclic diguanylate monophosphate (cdGMP) bypass this requirement. Multiple lines of evidence suggest that the UPP has a central polysaccharide component. Using an A. tumefaciens derivative with elevated cdGMP and mutationally disabled for other dispensable polysaccharides, a series of related genetic screens have identified a large number of genes involved in UPP biosynthesis, most of which are Wzx-Wzy-type polysaccharide biosynthetic components. Extensive analyses of UPP production in these mutants have revealed that the UPP is composed of two genetically, chemically, and spatially discrete forms of polysaccharide, and that each requires a specific Wzy-type polymerase. Other important biosynthetic, processing, and regulatory functions for UPP production are also revealed, some of which are common to both polysaccharides, and a subset of which are specific to each type. Many of the UPP genes identified are conserved among diverse rhizobia, whereas others are more lineage specific.

Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection.

Treatment of bacterial infections is becoming a serious clinical challenge due to the global dissemination of multidrug antibiotic resistance, necessitating the search for alternative treatments to disarm the virulence mechanisms underlying these infections. Uropathogenic Escherichia coli (UPEC) employs multiple chaperone-usher pathway pili tipped with adhesins with diverse receptor specificities to colonize various host tissues and habitats. For example, UPEC F9 pili specifically bind galactose or N-acetylgalactosamine epitopes on the kidney and inflamed bladder. Using X-ray structure-guided methods, virtual screening, and multiplex ELISA arrays, we rationally designed aryl galactosides and N-acetylgalactosaminosides that inhibit the F9 pilus adhesin FmlH. The lead compound, 29ß-NAc, is a biphenyl N-acetyl-ß-galactosaminoside with a Ki of ∼90 nM, representing a major advancement in potency relative to the characteristically weak nature of most carbohydrate-lectin interactions. 29ß-NAc binds tightly to FmlH by engaging the residues Y46 through edge-to-face π-stacking with its A-phenyl ring, R142 in a salt-bridge interaction with its carboxylate group, and K132 through water-mediated hydrogen bonding with its N-acetyl group. Administration of 29ß-NAc in a mouse urinary tract infection (UTI) model significantly reduced bladder and kidney bacterial burdens, and coadministration of 29ß-NAc and mannoside 4Z269, which targets the type 1 pilus adhesin FimH, resulted in greater elimination of bacteria from the urinary tract than either compound alone. Moreover, FmlH specifically binds healthy human kidney tissue in a 29ß-NAc-inhibitable manner, suggesting a key role for F9 pili in human kidney colonization. Thus, these glycoside antagonists of FmlH represent a rational antivirulence strategy for UPEC-mediated UTI treatment.

Discrete Responses to Limitation for Iron and Manganese in Agrobacterium tumefaciens: Influence on Attachment and Biofilm Formation.

UNLABELLED: Transition metals such as iron and manganese are crucial trace nutrients for the growth of most bacteria, functioning as catalytic cofactors for many essential enzymes. Dedicated uptake and regulatory systems have evolved to ensure their acquisition for growth, while preventing toxicity. Transcriptomic analysis of the iron- and manganese-responsive regulons of Agrobacterium tumefaciens revealed that there are discrete regulatory networks that respond to changes in iron and manganese levels. Complementing earlier studies, the iron-responsive gene network is quite large and includes many aspects of iron-dependent metabolism and the iron-sparing response. In contrast, the manganese-responsive network is restricted to a limited number of genes, many of which can be linked to transport and utilization of the transition metal. Several of the target genes predicted to drive manganese uptake are required for growth under manganese-limited conditions, and an A. tumefaciens mutant with a manganese transport deficiency is attenuated for plant virulence. Iron and manganese limitation independently inhibit biofilm formation by A. tumefaciens, and several candidate genes that could impact biofilm formation were identified in each regulon. The biofilm-inhibitory effects of iron and manganese do not rely on recognized metal-responsive transcriptional regulators, suggesting alternate mechanisms influencing biofilm formation. However, under low-manganese conditions the dcpA operon is upregulated, encoding a system that controls levels of the cyclic di-GMP second messenger. Mutation of this regulatory pathway dampens the effect of manganese limitation. IMPORTANCE: Responses to changes in transition metal levels, such as those of manganese and iron, are important for normal metabolism and growth in bacteria. Our study used global gene expression profiling to understand the response of the plant pathogen Agrobacterium tumefaciens to changes of transition metal availability. Among the properties that are affected by both iron and manganese levels are those required for normal surface attachment and biofilm formation, but the requirement for each of these transition metals is mechanistically independent from the other.

Inhibition and dispersal of Agrobacterium tumefaciens biofilms by a small diffusible Pseudomonas aeruginosa exoproduct(s).

Environmental biofilms often contain mixed populations of different species. In these dense communities, competition between biofilm residents for limited nutrients such as iron can be fierce, leading to the evolution of competitive factors that affect the ability of competitors to grow or form biofilms. We have discovered a compound(s) present in the conditioned culture fluids of Pseudomonas aeruginosa that disperses and inhibits the formation of biofilms produced by the facultative plant pathogen Agrobacterium tumefaciens. The inhibitory activity is strongly induced when P. aeruginosa is cultivated in iron-limited conditions, but it does not function through iron sequestration. In addition, the production of the biofilm inhibitory activity is not regulated by the global iron regulatory protein Fur, the iron-responsive extracytoplasmic function σ factor PvdS, or three of the recognized P. aeruginosa quorum-sensing systems. In addition, the compound(s) responsible for the inhibition and dispersal of A. tumefaciens biofilm formation is likely distinct from the recently identified P. aeruginosa dispersal factor, cis-2-decenoic acid (CDA), as dialysis of the culture fluids showed that the inhibitory compound was larger than CDA and culture fluids that dispersed and inhibited biofilm formation by A. tumefaciens had no effect on biofilm formation by P. aeruginosa.

Antiparallel and interlinked control of cellular iron levels by the Irr and RirA regulators of Agrobacterium tumefaciens.

The plant pathogen Agrobacterium tumefaciens encodes predicted iron-responsive regulators, Irr and RirA, that function in several other bacteria to control the response to environmental iron levels. Deletion mutations of irr and rirA, alone and in combination, were evaluated for their impact on cellular iron response. Growth was severely diminished in the Δirr mutant under iron-limiting conditions, but reversed to wild-type levels in an Δirr ΔrirA mutant. The level of uncomplexed iron in the Δirr mutant was decreased, whereas the ΔrirA mutant exhibited elevated iron levels. Sensitivity of the Δirr and ΔrirA mutants to iron-activated antimicrobial compounds generally reflected their uncomplexed-iron levels. Expression of genes that encode iron uptake systems was decreased in the Δirr mutant, whereas that of iron utilization genes was increased. Irr function required a trihistidine repeat likely to mediate interactions with heme. Iron uptake genes were derepressed in the ΔrirA mutant. In the Δirr ΔrirA mutant, iron uptake and utilization genes were derepressed, roughly combining the phenotypes of the single mutants. Siderophore production was elevated in the rirA mutant, but most strongly regulated by an RirA-controlled sigma factor. Expression of rirA itself was regulated by Irr, RirA, and iron availability, in contrast to irr expression, which was relatively stable in the different mutants. These studies suggest that in A. tumefaciens, the Irr protein is most active under low-iron conditions, inhibiting iron utilization and activating iron acquisition, while the RirA protein is active under high-iron conditions, repressing iron uptake.

Adaptation of Arginine Synthesis among Uropathogenic Branches of the Escherichia coli Phylogeny Reveals Adjustment to the Urinary Tract Habitat.

Urinary tract infections (UTIs) are predominantly caused by uropathogenic Escherichia coli (UPEC). UPEC pathogenesis requires passage through a severe population bottleneck involving intracellular bacterial communities (IBCs) that are clonal expansions of a single invading UPEC bacterium in a urothelial superficial facet cell. IBCs occur only during acute pathogenesis. The bacteria in IBCs form the founder population that develops into persistent extracellular infections. Only a small fraction of UPEC organisms proceed through the IBC cycle, regardless of the inoculum size. This dramatic reduction in population size precludes the utility of genomic mutagenesis technologies for identifying genes important for persistence. To circumvent this bottleneck, we previously identified 29 positively selected genes (PSGs) within UPEC and hypothesized that they contribute to virulence. Here, we show that 8 of these 29 PSGs are required for fitness during persistent bacteriuria. Conversely, 7/8 of these PSG mutants showed essentially no phenotype in acute UTI. Deletion of the PSG argI leads to arginine auxotrophy. Relative to the other arg genes, argI in the B2 clade (which comprises most UPEC strains) of E. coli has diverged from argI in other E. coli clades. Replacement of argI in a UPEC strain with a non-UPEC argI allele complemented the arginine auxotrophy but not the persistent bacteriuria defect, showing that the UPEC argI allele contributes to persistent infection. These results highlight the complex roles of metabolic pathways during infection and demonstrate that evolutionary approaches can identify infection-specific gene functions downstream of population bottlenecks, shedding light on virulence and the genetic evolution of pathogenesis.IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the most common cause of human urinary tract infection (UTI). Population bottlenecks during early stages of UTI make high-throughput screens impractical for understanding clinically important later stages of UTI, such as persistence and recurrence. As UPEC is hypothesized to be adapted to these later pathogenic stages, we previously identified 29 genes evolving under positive selection in UPEC. Here, we found that 8 of these genes, including argI (which is involved in arginine biosynthesis), are important for persistence in a mouse model of UTI. Deletion of argI and other arginine synthesis genes resulted in (i) arginine auxotrophy and (ii) defects in persistent UTI. Replacement of a B2 clade argI with a non-B2 clade argI complemented arginine auxotrophy, but the resulting strain remained attenuated in its ability to cause persistent bacteriuria. Thus, argI may have a second function during UTI that is not related to simple arginine synthesis. This study demonstrates how variation in metabolic genes can impact virulence and provides insight into the mechanisms and evolution of bacterial virulence.

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes.

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.

Host range and molecular phylogenies of the soft rot enterobacterial genera pectobacterium and dickeya.

ABSTRACT Pectobacterium and Dickeya spp. are related broad-host-range entero-bacterial pathogens of angiosperms. A review of the literature shows that these genera each cause disease in species from at least 35% of angiosperm plant orders. The known host ranges of these pathogens partially overlap and, together, these two genera are pathogens of species from 50% of angiosperm plant orders. Notably, there are no reported hosts for either genus in the eudicots clade and no reported Dickeya hosts in the magnoliids or eurosids II clades, although Pectobacterium spp. are pathogens of at least one plant species in the magnoliids and at least one in each of the three eurosids II plant orders. In addition, Dickeya but not Pectobacterium spp. have been reported on a host in the rosids clade and, unlike Pectobacterium spp., have been reported on many Poales species. Natural disease among nonangiosperms has not been reported for either genus. Phylogenetic analyses of sequences concatenated from regions of seven housekeeping genes (acnA, gapA, icdA, mdh, mtlD, pgi, and proA) from representatives of these genera demonstrated that Dickeya spp. and the related tree pathogens, the genus Brenneria, are more diverse than Pectobacterium spp. and that the Pectobacterium strains can be divided into at least five distinct clades, three of which contain strains from multiple host plants.

Evolutionary fine-tuning of conformational ensembles in FimH during host-pathogen interactions.

Positive selection in the two-domain type 1 pilus adhesin FimH enhances Escherichia coli fitness in urinary tract infection (UTI). We report a comprehensive atomic-level view of FimH in two-state conformational ensembles in solution, composed of one low-affinity tense (T) and multiple high-affinity relaxed (R) conformations. Positively selected residues allosterically modulate the equilibrium between these two conformational states, each of which engages mannose through distinct binding orientations. A FimH variant that only adopts the R state is severely attenuated early in a mouse model of uncomplicated UTI but is proficient at colonizing catheterized bladders in vivo or bladder transitional-like epithelial cells in vitro. Thus, the bladder habitat has barrier(s) to R state-mediated colonization possibly conferred by the terminally differentiated bladder epithelium and/or decoy receptors in urine. Together, our studies reveal the conformational landscape in solution, binding mechanisms, and adhesive strength of an allosteric two-domain adhesin that evolved "moderate" affinity to optimize persistence in the bladder during UTI.

Bacterial virulence phenotypes of Escherichia coli and host susceptibility determine risk for urinary tract infections.

Urinary tract infections (UTIs) are caused by uropathogenic Escherichia coli (UPEC) strains. In contrast to many enteric E. coli pathogroups, no genetic signature has been identified for UPEC strains. We conducted a high-resolution comparative genomic study using E. coli isolates collected from the urine of women suffering from frequent recurrent UTIs. These isolates were genetically diverse and varied in their urovirulence, that is, their ability to infect the bladder in a mouse model of cystitis. We found no set of genes, including previously defined putative urovirulence factors (PUFs), that were predictive of urovirulence. In addition, in some patients, the E. coli strain causing a recurrent UTI had fewer PUFs than the supplanted strain. In competitive experimental infections in mice, the supplanting strain was more efficient at colonizing the mouse bladder than the supplanted strain. Despite the lack of a clear genomic signature for urovirulence, comparative transcriptomic and phenotypic analyses revealed that the expression of key conserved functions during culture, such as motility and metabolism, could be used to predict subsequent colonization of the mouse bladder. Together, our findings suggest that UTI risk and outcome may be determined by complex interactions between host susceptibility and the urovirulence potential of diverse bacterial strains.

The unexplored relationship between urinary tract infections and the autonomic nervous system.

Urinary tract infections (UTIs), the majority of which are caused by uropathogenic E. coli (UPEC), are extremely common infections that preferentially effect women. Additional complicating factors, such as catheterization, diabetes, and spinal cord injuries can increase the frequency and severity of UTIs. The rise of antimicrobial resistant uropathogens and the ability of this disease to chronically recur make the development of alternative preventative and therapeutic modalities a priority. The major symptoms of UTIs, urgency, frequency, and dysuria, are readouts of the autonomic nervous system (ANS) and the majority of the factors that lead to complicated UTIs have been shown to impact ANS function. This review summarizes the decades' long efforts to understand the molecular mechanisms of the interactions between UPEC and the host, with a particular focus on the recent findings revealing the molecular, bacteriological, immunological and epidemiological complexity of pathogenesis. Additionally, we describe the progress that has been made in: i) generating vaccines and anti-virulence compounds that prevent and/or treat UTI by blocking bacterial adherence to urinary tract tissue and; and ii) elucidating the mechanism by which anti-inflammatories are able to alleviate symptoms and improve disease prognosis. Finally, the potential relationships between the ANS and UTI are considered throughout. While these relationships have not been experimentally explored, the known interactions between numerous UTI characteristics (symptoms, complicating factors, and inflammation) and ANS function suggest that UTIs are directly impacting ANS stimulation and that ANS (dys)function may alter UTI prognosis.

Metabolic Requirements of Escherichia coli in Intracellular Bacterial Communities during Urinary Tract Infection Pathogenesis.

UNLABELLED: Uropathogenic Escherichia coli (UPEC) is the primary etiological agent of over 85% of community-acquired urinary tract infections (UTIs). Mouse models of infection have shown that UPEC can invade bladder epithelial cells in a type 1 pilus-dependent mechanism, avoid a TLR4-mediated exocytic process, and escape into the host cell cytoplasm. The internalized UPEC can clonally replicate into biofilm-like intracellular bacterial communities (IBCs) of thousands of bacteria while avoiding many host clearance mechanisms. Importantly, IBCs have been documented in urine from women and children suffering acute UTI. To understand this protected bacterial niche, we elucidated the transcriptional profile of bacteria within IBCs using microarrays. We delineated the upregulation within the IBC of genes involved in iron acquisition, metabolism, and transport. Interestingly, lacZ was highly upregulated, suggesting that bacteria were sensing and/or utilizing a galactoside for metabolism in the IBC. A ΔlacZ strain displayed significantly smaller IBCs than the wild-type strain and was attenuated during competitive infection with a wild-type strain. Similarly, a galK mutant resulted in smaller IBCs and attenuated infection. Further, analysis of the highly upregulated gene yeaR revealed that this gene contributes to oxidative stress resistance and type 1 pilus production. These results suggest that bacteria within the IBC are under oxidative stress and, consistent with previous reports, utilize nonglucose carbon metabolites. Better understanding of the bacterial mechanisms used for IBC development and establishment of infection may give insights into development of novel anti-virulence strategies. IMPORTANCE: Urinary tract infections (UTIs) are one of the most common bacterial infections, impacting mostly women. Every year, millions of UTIs occur in the U.S. with most being caused by uropathogenic E. coli(UPEC). During a UTI, UPEC invade bladder cells and form an intracellular bacterial community (IBC) that allows for the bacteria to replicate protected from the host immune response. In this study, we investigated genes that are expressed by UPEC within the IBC and determined how they contribute to the formation of this specialized community. Our findings suggest that galactose is important for UPEC growth in the IBC. Additionally, we found that a gene involved in oxidative stress is also important in the regulation of a key factor needed for UPEC invasion of bladder cells. These results may open the door for the development of treatments to diminish UTI frequency and/or severity.

Human Urine Decreases Function and Expression of Type 1 Pili in Uropathogenic Escherichia coli.

UNLABELLED: Uropathogenic Escherichia coli (UPEC) is the primary cause of community-acquired urinary tract infections (UTIs). UPEC bind the bladder using type 1 pili, encoded by the fim operon in nearly all E. coli. Assembled type 1 pili terminate in the FimH adhesin, which specifically binds to mannosylated glycoproteins on the bladder epithelium. Expression of type 1 pili is regulated in part by phase-variable inversion of the genomic element containing the fimS promoter, resulting in phase ON (expressing) and OFF (nonexpressing) orientations. Type 1 pili are essential for virulence in murine models of UTI; however, studies of urine samples from human UTI patients demonstrate variable expression of type 1 pili. We provide insight into this paradox by showing that human urine specifically inhibits both expression and function of type 1 pili. Growth in urine induces the fimS phase OFF orientation, preventing fim expression. Urine also contains inhibitors of FimH function, and this inhibition leads to a further bias in fimS orientation toward the phase OFF state. The dual effect of urine on fimS regulation and FimH binding presents a potential barrier to type 1 pilus-mediated colonization and invasion of the bladder epithelium. However, FimH-mediated attachment to human bladder cells during growth in urine reverses these effects such that fim expression remains ON and/or turns ON. Interestingly, FimH inhibitors called mannosides also induce the fimS phase OFF orientation. Thus, the transduction of FimH protein attachment or inhibition into epigenetic regulation of type 1 pilus expression has important implications for the development of therapeutics targeting FimH function. IMPORTANCE: Urinary tract infections (UTIs) are extremely common infections, frequently caused by uropathogenic Escherichia coli (UPEC), that are treated with antibiotics but often recur. Therefore, UTI treatment both is complicated by and contributes to bacterial antibiotic resistance. Thus, it is important to understand UTI pathogenesis to devise novel strategies and targets for prevention and treatment. Based on evidence from disease epidemiology and mouse models of infection, UPEC relies heavily on type 1 pili to attach to and invade the bladder epithelium during initial stages of UTI. Here, we demonstrate that the negative effect of planktonic growth in human urine on both the function and expression of type 1 pili is overcome by attachment to bladder epithelial cells, representing a strategy to subvert this alternative innate defense mechanism. Furthermore, this dually inhibitory action of urine is a mechanism shared with recently developed anti-type 1 pilus molecules, highlighting the idea that further development of antivirulence strategies targeting pili may be particularly effective for UPEC.

Establishment and Characterization of UTI and CAUTI in a Mouse Model.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

Genomic diversity and fitness of E. coli strains recovered from the intestinal and urinary tracts of women with recurrent urinary tract infection.

Urinary tract infections (UTIs) are common in women, and recurrence is a major clinical problem. Most UTIs are caused by uropathogenic Escherichia coli (UPEC). UPEC are generally thought to migrate from the gut to the bladder to cause UTI. UPEC form specialized intracellular bacterial communities in the bladder urothelium as part of a pathogenic mechanism to establish a foothold during acute stages of infection. Evolutionarily, such a specific adaptation to the bladder environment would be predicted to result in decreased fitness in other habitats, such as the gut. To examine this prediction, we characterized 45 E. coli strains isolated from the feces and urine of four otherwise healthy women with recurrent UTI. Multilocus sequence typing and whole genome sequencing revealed that two patients maintained a clonal population in both these body habitats throughout their recurrent UTIs, whereas the other two exhibited a wholesale shift in the dominant UPEC strain colonizing both sites. In vivo competition studies in mouse models, using isolates taken from one of the patients with a wholesale population shift, revealed that the strain that dominated her last UTI episode had increased fitness in both the gut and the bladder relative to the strain that dominated in preceding episodes. Increased fitness correlated with differences in the strains' gene repertoires and carbohydrate and amino acid utilization profiles. Thus, UPEC appear capable of persisting in both the gut and urinary tract without a fitness trade-off, emphasizing the need to widen our consideration of potential reservoirs for strains causing recurrent UTI.

Bacterial competition: surviving and thriving in the microbial jungle.

Most natural environments harbour a stunningly diverse collection of microbial species. In these communities, bacteria compete with their neighbours for space and resources. Laboratory experiments with pure and mixed cultures have revealed many active mechanisms by which bacteria can impair or kill other microorganisms. In addition, a growing body of theoretical and experimental population studies indicates that the interactions within and between bacterial species can have a profound impact on the outcome of competition in nature. The next challenge is to integrate the findings of these laboratory and theoretical studies and to evaluate the predictions that they generate in more natural settings.