Bourret, T. J. | Search bioRxiv

bioRxiv2024

In vitro assays for clinical isolates of sequence type 131 Escherichia coli do not recapitulate in vivo infectivity using a murine model of urinary tract infection

Sequence Type 131 isolates are a major cause of cystitis and pyelonephritis. Many studies rely solely on in vitro assays to screen for bacterial virulence factors associated with the pathogenicity of clinical isolates of E. coli. Few studies have compared in vitro findings to in vivo infectivity of clinical isolates. The purpose of this study was to evaluate the correlation between in vitro assays with the ability to cause cystitis and pyelonephritis in a murine model of urinary tract infection. In vitro assays were conducted according to published protocols and included: motility assays, biofilm formation, epithelial cell adhesion and invasion, and curli production. Twenty-one UPEC isolates of E. coli ST131 and non-ST131 were used for both in vivo and in vitro studies. Six mice per isolate were inoculated via urethral catheterization. CFUs were determined from bladder and kidneys. In vitro and in vivo correlations were evaluated by multiple linear regression analysis. Pairwise linear regressions showed trendlines with weak positive correlations for motility, adhesion, and invasion and weak negative correlations for hemagglutination, biofilm and curli production. The ability of E. coli ST131 and non-ST131 clinical isolates to cause cystitis and pyelonephritis varies among strains. The R2 Pearson Correlation value was less than {+/-}0.5 for any pair, indicating little to no statistical association between in vitro and in vivo findings. These data show in vitro data are not predictive of the ability of ST131 E. coli to infect and/or cause disease in a mouse model. Author summaryUrinary tract infections affect 150 million people annually and E. coli ST131 have become the pandemic strain responsible for a majority of UTI, cystitis, and pyelonephritis cases. How ST131 E. coli have become such prolific strain still remains to be elucidated. When evaluating bacterial pathogenicity, it is customary practice to use in vitro assays to predict isolate virulence and mechanisms of fitness, due to the lower cost, and relative ease of experimentation compared to more costly and complicated in vivo models. It is also common to use model organisms like pathogenic E. coli CFT073 or non-pathogenic lab strains such as BW25113 as representatives for the entire species. However, our research has shown that not only are model organisms substantially different from clinical isolates of ST131 E. coli, but in vitro assays are poor predictors of clinical isolates ability to cause infection in a murine model of UTI. As such, research into the mechanisms of fitness for ST131 infectivity need to veer away from studying only model organisms and focus on utilizing pathogenic clinical isolates in conditions that more closely recapitulate urinary tract environmental niches.

microbiology↗

bioRxiv2020

DksA-dependent regulation of RpoS contributes to Borrelia burgdorferi tick-borne transmission and mammalian infectivity

Throughout its enzootic cycle, the Lyme disease spirochete Borreliella (Borrelia) burgdorferi, senses and responds to changes in its environment by using a small repertoire of transcription factors which coordinate the expression of genes required for infection of Ixodes ticks and various mammalian hosts. Among these transcription factors, the DnaK suppressor protein (DksA) plays a pivotal role in regulating gene expression in B. burgdorferi during periods of nutrient limitation and is required for mammalian infectivity. In many pathogenic bacteria, the gene regulatory activity of DksA along with the alarmone guanosine penta- and tetra-phosphate ((p)ppGpp) coordinates the stringent response to various environmental stresses including nutrient limitation. In this study, we sought to characterize the role of DksA in regulating the transcriptional activity of RNA polymerase and in the regulation of RpoS-dependent gene expression required for B. burgdorferi infectivity. Using in vitro transcription assays, we observed recombinant DksA inhibits RpoD-dependent transcription by B. burgdorferi RNA polymerase independent of ppGpp Additionally, we determined the pH-inducible expression of RpoS-dependent genes relies on DksA, but is independent of (p)ppGpp produced by Relbbu. Subsequent transcriptomic and western blot assays indicated DksA regulates the expression of BBD18, a protein previously implicated in the post-transcriptional regulation of RpoS. Moreover, we observed DksA was required for infection of mice following intraperitoneal inoculation or for transmission of B. burgdorferi by Ixodes scapularis nymphs. Together, these data suggest DksA plays a central role in coordinating transcriptional responses of B. burgdorferi required for infectivity through its interactions with RNA polymerase and post-transcriptional control of RpoS. Author SummaryLyme disease, caused by the spirochetal bacteria Borrelia burgdorferi, is the most common vector-borne illness in North America. The ability of B. burgdorferi to establish infection is predicated by its ability to coordinate the expression of virulence factors in response to diverse environmental stimuli encountered within Ixodes ticks and mammalian hosts. Previous studies have shown an essential role for the alternative sigma factor RpoS in regulating the expression of genes required for the successful transmission of B. burgdorferi by Ixodes ticks and infection of mammalian hosts. The DnaK suppressor protein (DksA) is a global gene regulator in B. burgdorferi that also contributes to the expression of RpoS-dependent genes. In this study, we determined DksA exerts its gene regulatory function through direct interactions with the B. burgdorferi RNA polymerase using in vitro transcription assays and controls the expression of RpoS-dependent genes required for mammalian infection by post-transcriptionally regulating cellular levels of RpoS. Our results demonstrate the utility of in vitro transcription assays to determine how gene regulatory proteins like DksA control gene expression in B. burgdorferi, and reveal a novel role for DksA in the infectious cycle of B. burgdorferi.

microbiology↗