Swaine L. Chen

Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle

Studies of the genetic network that controls the Caulobacter cell cycle have identified a response regulator, CtrA, that controls, directly or indirectly, one-quarter of the 553 cell cycle-regulated genes. We have performed in vivo genomic binding site analysis of the CtrA protein to identify which of these genes have regulatory regions bound directly by CtrA. By combining these data with previous global analysis of cell cycle transcription patterns and gene expression profiles of mutant ctrA strains, we have determined that CtrA directly regulates at least 95 genes. The total group of CtrA-regulated genes includes those involved in polar morphogenesis, DNA replication initiation, DNA methylation, cell division, and cell wall metabolism. Also among the genes in this notably large regulon are 14 that encode regulatory proteins, including 10 two-component signal transduction regulatory proteins. Identification of additional regulatory genes activated by CtrA will serve to directly connect new regulatory modules to the network controlling cell cycle progression.

Positive selection identifies an in vivo role for FimH during urinary tract infection in addition to mannose binding

FimH, the type 1 pilus adhesin of uropathogenic Escherichia coli (UPEC), contains a receptor-binding domain with an acidic binding pocket specific for mannose. The fim operon, and thus type 1 pilus production, is under transcriptional control via phase variation of an invertible promoter element. FimH is critical during urinary tract infection for mediating colonization and invasion of the bladder epithelium and establishment of intracellular bacterial communities (IBCs). In silico analysis of FimH gene sequences from 279 E. coli strains identified specific amino acids evolving under positive selection outside of its mannose-binding pocket. Mutating two of these residues (A27V/V163A) had no effect on phase variation, pilus assembly, or mannose binding in vitro. However, compared to wild-type, this double mutant strain exhibited a 10,000-fold reduction in mouse bladder colonization 24 h after inoculation and was unable to form IBCs even though it bound normally to mannosylated receptors in the urothelium. In contrast, the single A62S mutation altered phase variation, reducing the proportion of piliated cells, reduced mannose binding 8-fold, and decreased bladder colonization 30-fold in vivo compared to wild-type. A phase-locked ON A62S mutant restored virulence to wild-type levels even though in vitro mannose binding remained impaired. Thus, positive selection analysis of FimH has separated mannose binding from in vivo fitness, suggesting that IBC formation is critical for successful infection of the mammalian bladder, providing support for more general use of in silico positive selection analysis to define the molecular underpinnings of bacterial pathogenesis.

Population dynamics and niche distribution of uropathogenic Escherichia coli during acute and chronic urinary tract infection.

ABSTRACT Urinary tract infections (UTIs) have complex dynamics, with uropathogenic Escherichia coli (UPEC), the major causative agent, capable of colonization from the urethra to the kidneys in both extracellular and intracellular niches while also producing chronic persistent infections and frequent recurrent disease. In mouse and human bladders, UPEC invades the superficial epithelium, and some bacteria enter the cytoplasm to rapidly replicate into intracellular bacterial communities (IBCs) comprised of ∼104 bacteria each. Through IBC formation, UPEC expands in numbers while subverting aspects of the innate immune response. Within 12 h of murine bladder infection, half of the bacteria are intracellular, with 3 to 700 IBCs formed. Using mixed infections with green fluorescent protein (GFP) and wild-type (WT) UPEC, we discovered that each IBC is clonally derived from a single bacterium. Genetically tagged UPEC and a multiplex PCR assay were employed to investigate the distribution of UPEC throughout urinary tract niches over time. In the first 24 h postinfection (hpi), the fraction of tags dramatically decreased in the bladder and kidney, while the number of CFU increased. The percentage of tags detected at 6 hpi correlated to the number of IBCs produced, which closely matched a calculated multinomial distribution based on IBC clonality. The fraction of tags remaining thereafter depended on UTI outcome, which ranged from resolution of infection with or without quiescent intracellular reservoirs (QIRs) to the development of chronic cystitis as defined by persistent bacteriuria. Significantly more tags remained in mice that developed chronic cystitis, arguing that during the acute stages of infection, a higher number of IBCs precedes chronic cystitis than precedes QIR formation.

Fast and sensitive mapping of nanopore sequencing reads with GraphMap.

Realizing the democratic promise of nanopore sequencing requires the development of new bioinformatics approaches to deal with its specific error characteristics. Here we present GraphMap, a mapping algorithm designed to analyse nanopore sequencing reads, which progressively refines candidate alignments to robustly handle potentially high-error rates and a fast graph traversal to align long reads with speed and high precision (>95%). Evaluation on MinION sequencing data sets against short- and long-read mappers indicates that GraphMap increases mapping sensitivity by 10–80% and maps >95% of bases. GraphMap alignments enabled single-nucleotide variant calling on the human genome with increased sensitivity (15%) over the next best mapper, precise detection of structural variants from length 100 bp to 4 kbp, and species and strain-specific identification of pathogens using MinION reads. GraphMap is available open source under the MIT license at https://github.com/isovic/graphmap.

Helicobacter pylori evolution during progression from chronic atrophic gastritis to gastric cancer and its impact on gastric stem cells

We have characterized the adaptations of Helicobacter pylori to a rarely captured event in the evolution of its impact on host biology—the transition from chronic atrophic gastritis (ChAG) to gastric adenocarcinoma—and defined the impact of these adaptations on an intriguing but poorly characterized interaction between this bacterium and gastric epithelial stem cells. Bacterial isolates were obtained from a single human host colonized with a single dominant strain before and after his progression from ChAG to gastric adenocarcinoma during a 4-year interval. Draft genome assemblies were generated from two isolates, one ChAG-associated, the other cancer-associated. The cancer-associated strain was less fit in a gnotobiotic transgenic mouse model of human ChAG and better able to establish itself within a mouse gastric epithelial progenitor-derived cell line (mGEP) that supports bacterial attachment. GeneChip-based comparisons of the transcriptomes of mGEPs and a control mouse gastric epithelial cell line revealed that, upon infection, the cancer-associated strain regulates expression of GEP-associated signaling and metabolic pathways, and tumor suppressor genes associated with development of gastric cancer in humans, in a manner distinct from the ChAG-associated isolate. The effects on GEP metabolic pathways, some of which were confirmed in gnotobiotic mice, together with observed changes in the bacterial transcriptome are predicted to support aspects of an endosymbiosis between this microbe and gastric stem cells. These results provide insights about how H. pylori may adapt to and influence stem cell biology and how its intracellular residency could contribute to gastric tumorigenesis.

A central metabolic circuit controlled by QseC in pathogenic Escherichia coli

The QseC sensor kinase regulates virulence in multiple Gram‐negative pathogens, by controlling the activity of the QseB response regulator. We have previously shown that qseC deletion interferes with dephosphorylation of QseB thus unleashing what appears to be an uncontrolled positive feedback loop stimulating increased QseB levels. Deletion of QseC downregulates virulence gene expression and attenuates enterohaemorrhagic and uropathogenic Escherichia coli (EHEC and UPEC), Salmonella typhimurium, and Francisella tularensis. Given that these pathogens employ different infection strategies and virulence factors, we used genome‐wide approaches to better understand the role of the QseBC interplay in pathogenesis. We found that deletion of qseC results in misregulation of nucleotide, amino acid, and carbon metabolism. Comparable metabolic changes are seen in EHEC ΔqseC, suggesting that deletion of qseC confers similar pleiotropic effects in these two different pathogens. Disruption of representative metabolic enzymes phenocopied UPEC ΔqseC in vivo and resulted in virulence factor downregulation. We thus propose that in the absence of QseC, the constitutively active QseB leads to pleiotropic effects, impairing bacterial metabolism, and thereby attenuating virulence. These findings provide a basis for the development of antimicrobials targeting the phosphatase activity of QseC, as a means to attenuate a wide range of QseC‐bearing pathogens.

Diabetes Mellitus and Urinary Tract Infection: Epidemiology, Pathogenesis and Proposed Studies in Animal Models

PURPOSE We reviewed the current state of knowledge about urinary tract infection in patients with diabetes from the clinical and basic science perspectives. We identified key knowledge gaps and areas for further research. MATERIALS AND METHODS We performed a focused literature search on certain topics, including clinical studies related to etiology and pathophysiology of urinary tract infection in patients with diabetes, urinary tract infection studies in animal models of diabetes and basic science studies of the molecular mechanisms of urinary tract infection. RESULTS Individuals with diabetes are at higher risk for urinary tract infection. Increased susceptibility in patients with diabetes is positively associated with increased duration and severity of diabetes. Clinical epidemiological data identifying mechanisms of increased urinary tract infection susceptibility in patients with diabetes are generally lacking and indicate only that urinary tract infections in women with and without diabetes are qualitatively similar in bacterial etiology and morbid sequelae. Existing animal models for diabetes have not been well characterized for urinary tract infection research. The increased incidence, prevalence and severity of urinary tract infection in patients with diabetes argue for aggressive antibacterial chemotherapy but novel therapies resulting from urinary tract infection research in nondiabetic animal models are still not available. CONCLUSIONS Future clinical investigations of urinary tract infection in patients with diabetes should focus on how the disease differs from that in patients without diabetes, notably on the role of glycosuria and urinary tract infection risk. Basic science research priorities for urinary tract infection in patients with diabetes should emphasize further development of diabetic animal models for urinary tract infection research and clinical translation of known important virulence determinants into new therapies.

Genomic Diversity and Fitness of E. coli Strains Recovered from the Intestinal and Urinary Tracts of Women with Recurrent Urinary Tract Infection

Comparative genomic and functional studies reveal that E. coli strains from women with recurrent urinary tract infection can move between gut and urinary tract without a fitness trade-off. UTI Bugs Without Barriers Urinary tract infections (UTIs) are among the most common infections in women, with uropathogenic Escherichia coli (UPEC) being the major cause. Recurrent infections are troublesome and can persist for years. Studies in mice have led to the realization that UPEC strains can specialize so that once they enter the urinary tract they can invade bladder tissue, forming protected bacterial communities that contribute to recurrent UTIs. A prevailing view is that recurrent UTIs also represent repeated movement of UPEC strains from the gut to the bladder. This migration is thought to be unidirectional, reflecting a view that fitness (ability to succeed) in the bladder comes at a cost of loss of fitness in the gut. We reexamined this fitness “trade-off” by characterizing the genomes of urine and fecal E. coli isolates obtained from four healthy women, enrolled in a large patient study of recurrent UTI, who each had three recurrent UTIs. In two women, the dominant UPEC strain in both their urine and feces was the same throughout all three UTIs. In the other two, the UPEC strain present in both urine and feces in the initial UTI episode was replaced by a different strain at the third recurrence. In mouse models of bladder infection and gut colonization, the strain that dominated in the later UTI episode had increased fitness in both habitats compared to the strain it replaced. Increased fitness correlated with genetic differences affecting nutrient utilization and virulence. Thus, recurrent UTI is complex and may involve strains moving freely, without fitness trade-offs, between the bladder and the gut in addition to invasion of bladder tissue. Whereas further human studies are needed to assess the role of gut bacteria and their genetic characteristics during recurrent UTI, this broader view could lead to new approaches for prevention, diagnosis, and treatment of this troublesome 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.

LeuX tRNA-dependent and -independent mechanisms of Escherichia coli pathogenesis in acute cystitis.

Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity‐associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili‐mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI IIUTI89 disrupts the adjacent leuX tRNA locus. Loss of wild‐type leuX‐encoded tRNA5Leu significantly delayed, but did not eliminate, FimB recombinase‐mediated phase variation of type 1 pili. FimX, an additional FimB‐like, leuX‐independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non‐piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI IIUTI89 genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI IIUTI89 during the establishment of acute cystitis is maintenance of wild‐type leuX, and not PAI IIUTI89 gene content.

Positively selected FimH residues enhance virulence during urinary tract infection by altering FimH conformation.

Significance The evolution of multidrug resistance in pathogenic bacteria, including uropathogenic Escherichia coli (UPEC), that cause most urinary tract infections is becoming a worldwide crisis. UPEC use a variety of virulence factors and adhesins, including the mannose-binding FimH adhesin, to colonize and invade bladder tissue, often forming intracellular biofilms and quiescent reservoirs that can contribute to recurrent infections recalcitrant to treatment. Using two prototypical UPEC strains, we discovered that positively selected residues outside of the FimH mannose-binding pocket affect transitions between low- and high-affinity FimH conformations, which extraordinarily impacts FimH function during pathogenesis. Thus, this work elucidates mechanistic and functional insights into pathoadaptation and evolutionary fine-tuning of critical virulence interactions. Chaperone–usher pathway pili are a widespread family of extracellular, Gram-negative bacterial fibers with important roles in bacterial pathogenesis. Type 1 pili are important virulence factors in uropathogenic Escherichia coli (UPEC), which cause the majority of urinary tract infections (UTI). FimH, the type 1 adhesin, binds mannosylated glycoproteins on the surface of human and murine bladder cells, facilitating bacterial colonization, invasion, and formation of biofilm-like intracellular bacterial communities. The mannose-binding pocket of FimH is invariant among UPEC. We discovered that pathoadaptive alleles of FimH with variant residues outside the binding pocket affect FimH-mediated acute and chronic pathogenesis of two commonly studied UPEC strains, UTI89 and CFT073. In vitro binding studies revealed that, whereas all pathoadaptive variants tested displayed the same high affinity for mannose when bound by the chaperone FimC, affinities varied when FimH was incorporated into pilus tip-like, FimCGH complexes. Structural studies have shown that FimH adopts an elongated conformation when complexed with FimC, but, when incorporated into the pilus tip, FimH can adopt a compact conformation. We hypothesize that the propensity of FimH to adopt the elongated conformation in the tip corresponds to its mannose binding affinity. Interestingly, FimH variants, which maintain a high-affinity conformation in the FimCGH tip-like structure, were attenuated during chronic bladder infection, implying that FimH’s ability to switch between conformations is important in pathogenesis. Our studies argue that positively selected residues modulate fitness during UTI by affecting FimH conformation and function, providing an example of evolutionary tuning of structural dynamics impacting in vivo survival.