Mapping niche-specific two-component system requirements in uropathogenic Escherichia coli

ABSTRACT Sensory systems allow pathogens to differentiate between different niches and respond to stimuli within them. A major mechanism through which bacteria sense and respond to stimuli in their surroundings is two-component systems (TCSs). TCSs allow for the detection of multiple stimuli to lead to a highly controlled and rapid change in gene expression. Here, we provide a comprehensive list of TCSs important for the pathogenesis of uropathogenic Escherichia coli (UPEC). UPEC accounts for >75% of urinary tract infections (UTIs) worldwide. UTIs are most prevalent among people assigned female at birth, with the vagina becoming colonized by UPEC in addition to the gut and the bladder. In the bladder, adherence to the urothelium triggers E. coli invasion of bladder cells and an intracellular pathogenic cascade. Intracellular E. coli are safely hidden from host neutrophils, competition from the microbiota, and antibiotics that kill extracellular E. coli. To survive in these intimately connected, yet physiologically diverse niches E. coli must rapidly coordinate metabolic and virulence systems in response to the distinct stimuli encountered in each environment. We hypothesized that specific TCSs allow UPEC to sense these diverse environments encountered during infection with built-in redundant safeguards. Here, we created a library of isogenic TCS deletion mutants that we leveraged to map distinct TCS contributions to infection. We identify—for the first time—a comprehensive panel of UPEC TCSs that are critical for infection of the genitourinary tract and report that the TCSs mediating colonization of the bladder, kidneys, or vagina are distinct. IMPORTANCE While two-component system (TCS) signaling has been investigated at depth in model strains of Escherichia coli, there have been no studies to elucidate—at a systems level—which TCSs are important during infection by pathogenic Escherichia coli. Here, we report the generation of a markerless TCS deletion library in a uropathogenic E. coli (UPEC) isolate that can be leveraged for dissecting the role of TCS signaling in different aspects of pathogenesis. We use this library to demonstrate, for the first time in UPEC, that niche-specific colonization is guided by distinct TCS groups.

I found it surprising that none of the TCS mutants exhibited growth differences in vitro, particularly in minimal medium.Where there difference in density at which they achieved stationary phase?Have growth differences been found for any of these systems in other E. coli or Enterobacteriaceae?It would be helpful to include this information as part of the discussion.
The manuscript text appropriately qualifies the results by saying no defects were detected under the tested experimental conditions, and I do not feel that further testing of the full panel of mutants is necessary.However, the results would be strengthened by testing the mutants with in vivo bladder defects for growth in human urine.
The y-axis label of Figure 3D does not appear to correspond to the presented data.It would also be helpful to show the temporal urine CFU data as a supplemental figure.Reviewer #2 (Comments for the Author): This paper describes the construction of a complete set of two-component system knockouts in the urinary pathogenic E. coli UTI89.The various knockouts are characterized for growth in vitro, adherence to or invasion of urothelial cells, and colonization of the bladder, kidneys and vagina in a mouse UTI model.The authors find that different TCSs are important for colonization of different niches.The results highlight some systems associated with respiration.I believe the results will be of interest to the community studying UTIs and also to those interested more generally in host-microbe interactions and two-component signaling.In addition, the strain collection will be a great resource for this community.
I have only a few specific comments.1) Lines 217-218 and Fig. 1.The results indicate that UTI89 has a specific growth rate of about 0.2/hr in both LB and N-minimal medium.This result raises two questions: a) Why does UTI89 grow so slowly in LB at 37 degrees with shaking?A specific growth rate of 0.2/hr is a doubling time of about 3.5 hours, which is much longer than the behavior of most E. coli strains in LB; b) How is it possible that UTI89 has virtually the same growth rate in LB and a minimal medium?Is this N-minimal medium in fact a very rich medium?If so, then it should not be described as a minimal medium.These questions raise concerns that there was a problem with the growth measurements or data analysis, so they need to be addressed.
2) Related to the above comments, both a reference for N-minimal medium and the recipe, including carbon source and supplements, if any, should be in the methods section of the paper.
3) There are a number of typos in Table 1 ("Prescence", Changes is osmolarity", ...).Also, line 230 mentions kguRS but I do not see the names KguR or KguS in Table 1.4) Lines 257-261.DtorS showed significant colonization defects in Fig. 2C, but deletion of its partner response regulator, DtorR, did not show such a defect.Doesn't this run counter to the conclusion that the TorRS TCS contribues to survival within the niche (line 260)?Perhaps deleting torS leads to inappropriate activation of TorR or possibly TorS also phosphorylates another response regulator.This should be discussed or at least the summary conclusion in line 260 "these TCSs contribute to survlval within a niche" should be adjusted accordingly.5) Fig. 2 I found it confusing that the deletions were not in the same order in panels A, B, C. I suggest keeping the same order across all three panels and using colors to highlight the significant systems in the various panels.

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January 19, 2024 1st Revision -Editorial Decision
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