TnSmu1 is a functional integrative and conjugative element in Streptococcus mutans that when expressed causes growth arrest of host bacteria.

Integrative and conjugative elements (ICEs) are major drivers of horizontal gene transfer in bacteria. They mediate their own transfer from host cells (donors) to recipients and allow bacteria to acquire new phenotypes, including pathogenic and metabolic capabilities and drug resistances. Streptococcus mutans, a major causative agent of dental caries, contains a putative ICE, TnSmu1, integrated at the 3' end of a leucyl tRNA gene. We found that TnSmu1 is a functional ICE, containing all the genes necessary for ICE function. It excised from the chromosome and excision was stimulated by DNA damage. We identified the DNA junctions generated by excision of TnSmu1, defined the ends of the element, and detected the extrachromosomal circle. We found that TnSmu1 can transfer from S. mutans donors to recipients when co-cultured on solid medium. The presence of TnSmu1 in recipients inhibited successful acquisition of another copy and this inhibition was mediated, at least in part, by the likely transcriptional repressor encoded by the element. Using microscopy to track individual cells, we found that activation of TnSmu1 caused an arrest of cell growth. Our results demonstrate that TnSmu1 is a functional ICE that affects the biology of its host cells.

Activation of the integrative and conjugative element Tn916 causes growth arrest and death of host bacteria.

Integrative and conjugative elements (ICEs) serve as major drivers of bacterial evolution. These elements often confer some benefit to host cells, including antibiotic resistance, metabolic capabilities, or pathogenic determinants. ICEs can also have negative effects on host cells. Here, we investigated the effects of the ICE (conjugative transposon) Tn916 on host cells. Because Tn916 is active in a relatively small subpopulation of host cells, we developed a fluorescent reporter system for monitoring activation of Tn916 in single cells. Using this reporter, we found that cell division was arrested in cells of Bacillus subtilis and Enterococcus faecalis (a natural host for Tn916) that contained an activated (excised) Tn916. Furthermore, most of the cells with the activated Tn916 subsequently died. We also observed these phenotypes on the population level in B. subtilis utilizing a modified version of Tn916 that can be activated in the majority of cells. We identified two genes (orf17 and orf16) in Tn916 that were sufficient to cause growth defects in B. subtilis and identified a single gene, yqaR, that is in a defective phage (skin) in the B. subtilis chromosome that was required for this phenotype. These three genes were only partially responsible for the growth defect caused by Tn916, indicating that Tn916 possesses multiple mechanisms to affect growth and viability of host cells. These results highlight the complex relationships that conjugative elements have with their host cells and the interplay between mobile genetic elements.

GAPDH mediates drug resistance and metabolism in Plasmodium falciparum malaria parasites.

Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Identifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH in MEP pathway control and antimalarial sensitivity.

Sex Differences in Population Dynamics during Formation of Kidney Bacterial Communities by Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC), the primary etiologic agent of urinary tract infections (UTIs), encounters a restrictive population bottleneck within the female mammalian bladder. Its genetic diversity is restricted during establishment of cystitis because successful UPEC must invade superficial bladder epithelial cells prior to forming clonal intracellular bacterial communities (IBCs). In this study, we aimed to understand UPEC population dynamics during ascending pyelonephritis, namely, formation of kidney bacterial communities (KBCs) in the renal tubular lumen and nucleation of renal abscesses. We inoculated the bladders of both male and female C3H/HeN mice, a background which features vesicoureteral reflux; we have previously shown that in this model, males develop severe, high-titer pyelonephritis and renal abscesses much more frequently than females. Mice were infected with 40 isogenic, PCR-tagged ("barcoded") UPEC strains, and tags remaining in bladder and kidneys were ascertained at intervals following infection. In contrast to females, males maintained a majority of strains within both the bladder and kidneys throughout the course of infection, indicating only a modest host-imposed bottleneck on overall population diversity during successful renal infection. Moreover, the diverse population in the infected male kidneys obscured any restrictive bottleneck in the male bladder. Finally, using RNA in situ hybridization following mixed infections with isogenic UPEC bearing distinct markers, we found that despite their extracellular location (in the urinary space), KBCs are clonal in origin. This finding indicates that even with bulk reflux of infected bladder urine into the renal pelvis, successful ascension of UPEC to establish the tubular niche is an uncommon event.

A host receptor enables type 1 pilus-mediated pathogenesis of Escherichia coli pyelonephritis.

Type 1 pili have long been considered the major virulence factor enabling colonization of the urinary bladder by uropathogenic Escherichia coli (UPEC). The molecular pathogenesis of pyelonephritis is less well characterized, due to previous limitations in preclinical modeling of kidney infection. Here, we demonstrate in a recently developed mouse model that beyond bladder infection, type 1 pili also are critical for establishment of ascending pyelonephritis. Bacterial mutants lacking the type 1 pilus adhesin (FimH) were unable to establish kidney infection in male C3H/HeN mice. We developed an in vitro model of FimH-dependent UPEC binding to renal collecting duct cells, and performed a CRISPR screen in these cells, identifying desmoglein-2 as a primary renal epithelial receptor for FimH. The mannosylated extracellular domain of human DSG2 bound directly to the lectin domain of FimH in vitro, and introduction of a mutation in the FimH mannose-binding pocket abolished binding to DSG2. In infected C3H/HeN mice, type 1-piliated UPEC and Dsg2 were co-localized within collecting ducts, and administration of mannoside FIM1033, a potent small-molecule inhibitor of FimH, significantly attenuated bacterial loads in pyelonephritis. Our results broaden the biological importance of FimH, specify the first renal FimH receptor, and indicate that FimH-targeted therapeutics will also have application in pyelonephritis.

Androgen exposure potentiates formation of intratubular communities and renal abscesses by Escherichia coli.

Females across their lifespan and certain male populations are susceptible to urinary tract infections (UTI). The influence of female vs. male sex on UTI is incompletely understood, in part because preclinical modeling has been performed almost exclusively in female mice. Here, we employed established and new mouse models of UTI with uropathogenic Escherichia coli (UPEC) to investigate androgen influence on UTI pathogenesis. Susceptibility to UPEC UTI in both male and female hosts was potentiated with 5α-dihydrotestosterone, while males with androgen receptor deficiency and androgenized females treated with the androgen receptor antagonist enzalutamide were protected from severe pyelonephritis. In androgenized females and in males, UPEC formed dense intratubular, biofilm-like communities, some of which were sheltered from infiltrating leukocytes by the tubular epithelium and by peritubular fibrosis. Abscesses were nucleated by small intratubular collections of UPEC first visualized at five days postinfection and briskly expanded over the subsequent 24 hours. Male mice deficient in Toll-like receptor 4, which fail to contain UPEC within abscesses, were susceptible to lethal dissemination. Thus, androgen receptor activation imparts susceptibility to severe upper-tract UTI in both female and male murine hosts. Visualization of intratubular UPEC communities illuminates early renal abscess pathogenesis and the role of abscess formation in preventing dissemination of infection. Additionally, our study suggests that androgen modulation may represent a novel therapeutic route to combat recalcitrant or recurrent UTI in a range of patient populations.

Dual Roles of Fer Kinase Are Required for Proper Hematopoiesis and Vascular Endothelium Organization during Zebrafish Development.

Fer kinase, a protein involved in the regulation of cell-cell adhesion and proliferation, has been shown to be required during invertebrate development and has been implicated in leukemia, gastric cancer, and liver cancer. However, in vivo roles for Fer during vertebrate development have remained elusive. In this study, we bridge the gap between the invertebrate and vertebrate realms by showing that Fer kinase is required during zebrafish embryogenesis for normal hematopoiesis and vascular organization with distinct kinase dependent and independent functions. In situ hybridization, quantitative PCR and fluorescence activated cell sorting (FACS) analyses revealed an increase in both erythrocyte numbers and gene expression patterns as well as a decrease in the organization of vasculature endothelial cells. Furthermore, rescue experiments have shown that the regulation of hematopoietic proliferation is dependent on Fer kinase activity, while vascular organizing events only require Fer in a kinase-independent manner. Our data suggest a model in which separate kinase dependent and independent functions of Fer act in conjunction with Notch activity in a divergent manner for hematopoietic determination and vascular tissue organization.

Renal scar formation and kidney function following antibiotic-treated murine pyelonephritis.

We present a new preclinical model to study treatment, resolution and sequelae of severe ascending pyelonephritis. Urinary tract infection (UTI), primarily caused by uropathogenic Escherichia coli (UPEC), is a common disease in children. Severe pyelonephritis is the primary cause of acquired renal scarring in childhood, which may eventually lead to hypertension and chronic kidney disease in a small but important fraction of patients. Preclinical modeling of UTI utilizes almost exclusively females, which (in most mouse strains) exhibit inherent resistance to severe ascending kidney infection; consequently, no existing preclinical model has assessed the consequences of recovery from pyelonephritis following antibiotic treatment. We recently published a novel mini-surgical bladder inoculation technique, with which male C3H/HeN mice develop robust ascending pyelonephritis, highly prevalent renal abscesses and evidence of fibrosis. Here, we devised and optimized an antibiotic treatment strategy within this male model to more closely reflect the clinical course of pyelonephritis. A 5-day ceftriaxone regimen initiated at the onset of abscess development achieved resolution of bladder and kidney infection. A minority of treated mice displayed persistent histological abscess at the end of treatment, despite microbiological cure of pyelonephritis; a matching fraction of mice 1 month later exhibited renal scars featuring fibrosis and ongoing inflammatory infiltrates. Successful antibiotic treatment preserved renal function in almost all infected mice, as assessed by biochemical markers 1 and 5 months post-treatment; hydronephrosis was observed as a late effect of treated pyelonephritis. An occasional mouse developed chronic kidney disease, generally reflecting the incidence of this late sequela in humans. In total, this model offers a platform to study the molecular pathogenesis of pyelonephritis, response to antibiotic therapy and emergence of sequelae, including fibrosis and renal scarring. Future studies in this system may inform adjunctive therapies that may reduce the long-term complications of this very common bacterial infection.

Urinary Tract Infection: Pathogenesis and Outlook.

The clinical syndromes comprising urinary tract infection (UTI) continue to exert significant impact on millions of patients worldwide, most of whom are otherwise healthy women. Antibiotic therapy for acute cystitis does not prevent recurrences, which plague up to one fourth of women after an initial UTI. Rising antimicrobial resistance among uropathogenic bacteria further complicates therapeutic decisions, necessitating new approaches based on fundamental biological investigation. In this review, we highlight contemporary advances in the field of UTI pathogenesis and how these might inform both our clinical perspective and future scientific priorities.