Transcriptome of Proteus mirabilis in the Murine Urinary Tract: Virulence and Nitrogen Assimilation Gene Expression

ABSTRACTThe enteric bacteriumProteus mirabilisis a common cause of complicated urinary tract infections. In this study, microarrays were used to analyzeP. mirabilisgene expressionin vivofrom experimentally infected mice. Urine was collected at 1, 3, and 7 days postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across nine microarrays, 471 genes were upregulated and 82 were downregulatedin vivocompared toin vitrobroth culture. Genes upregulatedin vivoencoded mannose-resistantProteus-like (MR/P) fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism enzymes, and a portion of the tricarboxylic acid (TCA) cycle enzymes. Flagella were downregulated. Ammonia assimilation geneglnA(glutamine synthetase) was repressedin vivo, whilegdhA(glutamate dehydrogenase) was upregulatedin vivo. Contrary to our expectations, ammonia availability due to urease activity inP. mirabilisdid not drive this gene expression. AgdhAmutant was growth deficient in minimal medium with citrate as the sole carbon source, and loss ofgdhAresulted in a significant fitness defect in the mouse model of urinary tract infection. UnlikeEscherichia coli, which repressesgdhAand upregulatesglnAin vivoand cannot utilize citrate, the data suggest thatP. mirabilisuses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential ofP. mirabilisin the urinary tract.

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New Aspects of RpoE in Uropathogenic Proteus mirabilis

Infection and Immunity ◽

10.1128/iai.02232-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 966-977 ◽

Cited By ~ 10

Author(s):

Ming-Che Liu ◽

Kuan-Ting Kuo ◽

Hsiung-Fei Chien ◽

Yi-Lin Tsai ◽

Shwu-Jen Liaw

Keyword(s):

Urinary Tract ◽

Virulence Factors ◽

Stress Responses ◽

Proteus Mirabilis ◽

Immune Cell ◽

Polymyxin B ◽

Urothelial Cells ◽

Cationic Antimicrobial Peptide ◽

Content Type ◽

Tract Infections

Proteus mirabilisis a common human pathogen causing recurrent or persistent urinary tract infections (UTIs). The underlying mechanisms forP. mirabilisto establish UTIs are not fully elucidated. In this study, we showed that loss of the sigma factor E (RpoE), mediating extracytoplasmic stress responses, decreased fimbria expression, survival in macrophages, cell invasion, and colonization in mice but increased the interleukin-8 (IL-8) expression of urothelial cells and swarming motility. This is the first study to demonstrate that RpoE modulated expression of MR/P fimbriae by regulatingmrpI, a gene encoding a recombinase controlling the orientation of MR/P fimbria promoter. By real-time reverse transcription-PCR, we found that the IL-8 mRNA amount of urothelial cells was induced significantly by lipopolysaccharides extracted fromrpoEmutant but not from the wild type. These RpoE-associated virulence factors should be coordinately expressed to enhance the fitness ofP. mirabilisin the host, including the avoidance of immune attacks. Accordingly,rpoEmutant-infected mice displayed more immune cell infiltration in bladders and kidneys during early stages of infection, and therpoEmutant had a dramatically impaired ability of colonization. Moreover, it is noteworthy that urea (the major component in urine) and polymyxin B (a cationic antimicrobial peptide) can induce expression ofrpoEby the reporter assay, suggesting that RpoE might be activated in the urinary tract. Altogether, our results indicate that RpoE is important in sensing environmental cues of the urinary tract and subsequently triggering the expression of virulence factors, which are associated with the fitness ofP. mirabilis, to build up a UTI.

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Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00157-20 ◽

2020 ◽

Vol 202 (20) ◽

Author(s):

Eric C. DiBiasio ◽

Hilary J. Ranson ◽

James R. Johnson ◽

David C. Rowley ◽

Paul S. Cohen ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Recurrent Infection ◽

Colony Growth ◽

Quiescent State ◽

Content Type ◽

Tract Infections

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling. IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

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The Pathogenic Potential of Proteus mirabilis Is Enhanced by Other Uropathogens during Polymicrobial Urinary Tract Infection

Infection and Immunity ◽

10.1128/iai.00808-16 ◽

2016 ◽

Vol 85 (2) ◽

Cited By ~ 36

Author(s):

Chelsie E. Armbruster ◽

Sara N. Smith ◽

Alexandra O. Johnson ◽

Valerie DeOrnellas ◽

Kathryn A. Eaton ◽

...

Keyword(s):

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Disease Severity ◽

Proteus Mirabilis ◽

Tissue Damage ◽

Urease Activity ◽

Pathogenic Potential ◽

Content Type

ABSTRACT Urinary catheter use is prevalent in health care settings, and polymicrobial colonization by urease-positive organisms, such as Proteus mirabilis and Providencia stuartii, commonly occurs with long-term catheterization. We previously demonstrated that coinfection with P. mirabilis and P. stuartii increased overall urease activity in vitro and disease severity in a model of urinary tract infection (UTI). In this study, we expanded these findings to a murine model of catheter-associated UTI (CAUTI), delineated the contribution of enhanced urease activity to coinfection pathogenesis, and screened for enhanced urease activity with other common CAUTI pathogens. In the UTI model, mice coinfected with the two species exhibited higher urine pH values, urolithiasis, bacteremia, and more pronounced tissue damage and inflammation compared to the findings for mice infected with a single species, despite having a similar bacterial burden within the urinary tract. The presence of P. stuartii, regardless of urease production by this organism, was sufficient to enhance P. mirabilis urease activity and increase disease severity, and enhanced urease activity was the predominant factor driving tissue damage and the dissemination of both organisms to the bloodstream during coinfection. These findings were largely recapitulated in the CAUTI model. Other uropathogens also enhanced P. mirabilis urease activity in vitro, including recent clinical isolates of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We therefore conclude that the underlying mechanism of enhanced urease activity may represent a widespread target for limiting the detrimental consequences of polymicrobial catheter colonization, particularly by P. mirabilis and other urease-positive bacteria.

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(p)ppGpp and CodY Promote Enterococcus faecalis Virulence in a Murine Model of Catheter-Associated Urinary Tract Infection

mSphere ◽

10.1128/msphere.00392-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 8

Author(s):

C. Colomer-Winter ◽

A. L. Flores-Mireles ◽

S. Kundra ◽

S. J. Hultgren ◽

J. A. Lemos

Keyword(s):

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Enterococcus Faecalis ◽

Stringent Response ◽

Nutrient Sensing ◽

Transcriptional Analysis ◽

Content Type ◽

Tract Infections

ABSTRACT In Firmicutes, the nutrient-sensing regulators (p)ppGpp, the effector molecule of the stringent response, and CodY work in tandem to maintain bacterial fitness during infection. Here, we tested (p)ppGpp and codY mutant strains of Enterococcus faecalis in a catheter-associated urinary tract infection (CAUTI) mouse model and used global transcriptional analysis to investigate the relationship of (p)ppGpp and CodY. The absence of (p)ppGpp or single inactivation of codY led to lower bacterial loads in catheterized bladders and diminished biofilm formation on fibrinogen-coated surfaces under in vitro and in vivo conditions. Single inactivation of the bifunctional (p)ppGpp synthetase/hydrolase rel did not affect virulence, supporting previous evidence that the association of (p)ppGpp with enterococcal virulence is not dependent on the activation of the stringent response. Inactivation of codY in the (p)ppGpp0 strain restored E. faecalis virulence in the CAUTI model as well as the ability to form biofilms in vitro. Transcriptome analysis revealed that inactivation of codY restores, for the most part, the dysregulated metabolism of (p)ppGpp0 cells. While a clear linkage between (p)ppGpp and CodY with expression of virulence factors could not be established, targeted transcriptional analysis indicates that a possible association between (p)ppGpp and c-di-AMP signaling pathways in response to the conditions found in the bladder may play a role in enterococcal CAUTI. Collectively, data from this study identify the (p)ppGpp-CodY network as an important contributor to enterococcal virulence in catheterized mouse bladder and support that basal (p)ppGpp pools and CodY promote virulence through maintenance of a balanced metabolism under adverse conditions. IMPORTANCE Catheter-associated urinary tract infections (CAUTIs) are one of the most frequent types of infection found in the hospital setting that can develop into serious and potentially fatal bloodstream infections. One of the infectious agents that frequently causes complicated CAUTI is the bacterium Enterococcus faecalis, a leading cause of hospital-acquired infections that are often difficult to treat due to the exceptional multidrug resistance of some isolates. Understanding the mechanisms by which E. faecalis causes CAUTI will aid in the discovery of new druggable targets to treat these infections. In this study, we report the importance of two nutrient-sensing bacterial regulators, named (p)ppGpp and CodY, for the ability of E. faecalis to infect the catheterized bladder of mice.

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In Vivo mRNA Profiling of Uropathogenic Escherichia coli from Diverse Phylogroups Reveals Common and Group-Specific Gene Expression Profiles

mBio ◽

10.1128/mbio.01075-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 39

Author(s):

Piotr Bielecki ◽

Uthayakumar Muthukumarasamy ◽

Denitsa Eckweiler ◽

Agata Bielecka ◽

Sarah Pohl ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Specific Gene ◽

Content Type ◽

E Coli ◽

Human Host ◽

Tract Infections

ABSTRACTmRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression ofEscherichia colipathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associatedE. coliisolates to different phylogenetic groups. Whereas thein vivogene expression profiles of the majority of genes were conserved among 21E. colistrains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribedin vivorelative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease.IMPORTANCEUrinary tract infections (UTI) are very common; at least half of all women experience UTI, most of which are caused by pathogenicEscherichia colistrains. In this study, we applied massive parallel cDNA sequencing (RNA-seq) to provide unbiased, deep, and accurate insight into the nature and the dimension of the uropathogenicE. coligene expression profile during an acute UTI within the human host. This work was undertaken to identify key players in physiological adaptation processes and, hence, potential targets for new infection prevention and therapy interventions specifically aimed at sabotaging bacterial adaptation to the human host.

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Role of Urinary Cathelicidin LL-37 and Human β-Defensin 1 in Uncomplicated Escherichia coli Urinary Tract Infections

Infection and Immunity ◽

10.1128/iai.01393-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1572-1578 ◽

Cited By ~ 41

Author(s):

Karen L. Nielsen ◽

Pia Dynesen ◽

Preben Larsen ◽

Lotte Jakobsen ◽

Paal S. Andersen ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Adult Women ◽

Content Type ◽

E Coli ◽

Innate Defense ◽

High Level ◽

Tract Infections

ABSTRACTCathelicidin (LL-37) and human β-defensin 1 (hBD-1) are important components of the innate defense in the urinary tract. The aim of this study was to characterize whether these peptides are important for developing uncomplicatedEscherichia coliurinary tract infections (UTIs). This was investigated by comparing urinary peptide levels of UTI patients during and after infection to those of controls, as well as characterizing the fecal flora of participants with respect to susceptibility to LL-37 andin vivovirulence. Forty-seven UTI patients and 50 controls who had never had a UTI were included. Participants were otherwise healthy, premenopausal, adult women. LL-37 MIC levels were compared for fecalE. coliclones from patients and controls and were also compared based on phylotypes (A, B1, B2, and D).In vivovirulence was investigated in the murine UTI model by use of selected fecal isolates from patients and controls. On average, UTI patients had significantly more LL-37 in urine during infection than postinfection, and patient LL-37 levels postinfection were significantly lower than those of controls. hBD-1 showed similar urine levels for UTI patients and controls. FecalE. coliisolates from controls had higher LL-37 susceptibility than fecal and UTIE. coliisolates from UTI patients.In vivostudies showed a high level of virulence of fecalE. coliisolates from both patients and controls and showed no difference in virulence correlated with the LL-37 MIC level. The results indicate that the concentration of LL-37 in the urinary tract and low susceptibility to LL-37 may increase the likelihood of UTI in a complex interplay between host and pathogen attributes.

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A Conserved PapB Family Member, TosR, Regulates Expression of the Uropathogenic Escherichia coli RTX Nonfimbrial Adhesin TosA while Conserved LuxR Family Members TosE and TosF Suppress Motility

Infection and Immunity ◽

10.1128/iai.01608-14 ◽

2014 ◽

Vol 82 (9) ◽

pp. 3644-3656 ◽

Cited By ~ 6

Author(s):

Michael D. Engstrom ◽

Christopher J. Alteri ◽

Harry L. T. Mobley

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Virulence Factors ◽

Upper Urinary Tract ◽

Host Cells ◽

Promoter Regions ◽

Content Type ◽

Tract Infections

ABSTRACTA heterogeneous subset of extraintestinal pathogenicEscherichia coli(ExPEC) strains, referred to as uropathogenicE. coli(UPEC), causes most uncomplicated urinary tract infections. However, no core set of virulence factors exists among UPEC strains. Instead, the focus of the analysis of urovirulence has shifted to studying broad classes of virulence factors and the interactions between them. For example, the RTX nonfimbrial adhesin TosA mediates adherence to host cells derived from the upper urinary tract. The associatedtosoperon is well expressedin vivobut poorly expressedin vitroand encodes TosCBD, a predicted type 1 secretion system. TosR and TosEF are PapB and LuxR family transcription factors, respectively; however, no role has been assigned to these potential regulators. Thus, the focus of this study was to determine how TosR and TosEF regulatetosAand affect the reciprocal expression of adhesins and flagella. Among a collection of sequenced UPEC strains, 32% (101/317) were found to encode TosA, and nearly all strains (91% [92/101]) simultaneously carried the putative regulatory genes. Deletion oftosRalleviatestosArepression. Thetospromoter was localized upstream oftosRusing transcriptional fusions of putative promoter regions withlacZ. TosR binds to this region, affecting a gel shift. A 100-bp fragment 220 to 319 bp upstream oftosRinhibits binding, suggesting localization of the TosR binding site. TosEF, on the other hand, downmodulate motility when overexpressed by preventing the expression offliC, encoding flagellin. Deletion oftosEFincreased motility. Thus, we present an additional example of the reciprocal control of adherence and motility.

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Identification of virulence determinants in uropathogenic Proteus mirabilis using signature-tagged mutagenesis

Journal of Medical Microbiology ◽

10.1099/jmm.0.2008/002071-0 ◽

2008 ◽

Vol 57 (9) ◽

pp. 1068-1078 ◽

Cited By ~ 38

Author(s):

Stephanie D. Himpsl ◽

C. Virginia Lockatell ◽

J. Richard Hebel ◽

David E. Johnson ◽

Harry L. T. Mobley

Keyword(s):

Urinary Tract ◽

Mouse Model ◽

Proteus Mirabilis ◽

Virulence Determinants ◽

Indwelling Catheters ◽

Cba Mice ◽

Transposon Mutants ◽

Tract Infections

The Gram-negative bacterium Proteus mirabilis causes urinary tract infections (UTIs) in individuals with long-term indwelling catheters or those with functional or structural abnormalities of the urinary tract. Known virulence factors include urease, haemolysin, fimbriae, flagella, DsbA, a phosphate transporter and genes involved in cell-wall synthesis and metabolism, many of which have been identified using the technique of signature-tagged mutagenesis (STM). To identify additional virulence determinants and to increase the theoretical coverage of the genome, this study generated and assessed 1880 P. mirabilis strain HI4320 mutants using this method. Mutants with disruptions in genes vital for colonization of the CBA mouse model of ascending UTI were identified after performing primary and secondary in vivo screens in approximately 315 CBA mice, primary and secondary in vitro screens in both Luria broth and minimal A medium to eliminate mutants with minor growth deficiencies, and co-challenge competition experiments in approximately 500 CBA mice. After completion of in vivo screening, a total of 217 transposon mutants were attenuated in the CBA mouse model of ascending UTI. Following in vitro screening, this number was reduced to 196 transposon mutants with a probable role in virulence. Co-challenge competition experiments confirmed significant attenuation for 37 of the 93 transposon mutants tested, being outcompeted by wild-type HI4320. Following sequence analysis of the 37 mutants, transposon insertions were identified in genes including the peptidyl-prolyl isomerases surA and ppiA, glycosyltransferase cpsF, biopolymer transport protein exbD, transcriptional regulator nhaR, one putative fimbrial protein, flagellar M-ring protein fliF and hook protein flgE, and multiple metabolic genes.

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Inhibition of Escherichia coli CFT073fliCExpression and Motility by Cranberry Materials

Applied and Environmental Microbiology ◽

10.1128/aem.05561-11 ◽

2011 ◽

Vol 77 (19) ◽

pp. 6852-6857 ◽

Cited By ~ 54

Author(s):

Gabriela Hidalgo ◽

Michelle Chan ◽

Nathalie Tufenkji

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Upper Urinary Tract ◽

Upec Strain ◽

Microscopy Imaging ◽

Content Type ◽

Reverse Transcription Pcr ◽

Strain Cft073 ◽

Tract Infections

ABSTRACTIn humans, uropathogenicEscherichia coli(UPEC) is the most common etiological agent of uncomplicated urinary tract infections (UTIs). Cranberry extracts have been linked to the prevention of UTIs for over a century; however, a mechanistic understanding of the way in which cranberry derivatives prevent bacterial infection is still lacking. In this study, we used afliC-luxreporter as well as quantitative reverse transcription-PCR to demonstrate that when UPEC strain CFT073 was grown or exposed to dehydrated, crushed cranberries or to purified cranberry-derived proanthocyanidins (cPACs), expression of the flagellin gene (fliC) was inhibited. In agreement with these results, transmission electron microscopy imaging of bacteria grown in the presence of cranberry materials revealed fewer flagella than those in bacteria grown under control conditions. Furthermore, we showed that swimming and swarming motilities were hindered when bacteria were grown in the presence of the cranberry compounds. Because flagellum-mediated motility has been suggested to enable UPEC to disseminate to the upper urinary tract, we propose that inhibition of flagellum-mediated motility might be a key mechanism by which cPACs prevent UTIs. This is the first report to show that cranberry compounds inhibit UPEC motility via downregulation of thefliCgene. Further studies are required to establish whether these inhibitors play a rolein vivo.

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Vaccination with Proteus Toxic Agglutinin, a Hemolysin-Independent Cytotoxin In Vivo, Protects against Proteus mirabilis Urinary Tract Infection

Infection and Immunity ◽

10.1128/iai.01050-08 ◽

2008 ◽

Vol 77 (2) ◽

pp. 632-641 ◽

Cited By ~ 34

Author(s):

Praveen Alamuri ◽

Kathryn A. Eaton ◽

Stephanie D. Himpsl ◽

Sara N. Smith ◽

Harry L. T. Mobley

Keyword(s):

Urinary Tract ◽

Proteus Mirabilis ◽

Parent Strain ◽

Double Mutant ◽

Vaccine Development ◽

Histopathological Examination ◽

Bacterial Load ◽

Passenger Domain ◽

Tract Infections

ABSTRACT Complicated urinary tract infections (UTI) caused by Proteus mirabilis are associated with severe pathology in the bladder and kidney. To investigate the roles of two established cytotoxins, the HpmA hemolysin, a secreted cytotoxin, and proteus toxic agglutinin (Pta), a surface-associated cytotoxin, mutant analysis was used in conjunction with a mouse model of ascending UTI. Inactivation of pta, but not inactivation of hpmA, resulted in significant decreases in the bacterial loads of the mutant in kidneys (P < 0.01) and spleens (P < 0.05) compared to the bacterial loads of the wild type; the 50% infective dose (ID50) of an isogenic pta mutant or hpmA pta double mutant was 100-fold higher (5 × 108 CFU) than the ID50 of parent strain HI4320 (5 × 106 CFU). Colonization by the parent strain caused severe cystitis and interstitial nephritis as determined by histopathological examination. Mice infected with the same bacterial load of the hpmA pta double mutant showed significantly reduced pathology (P < 0.01), suggesting that the additive effect of these two cytotoxins is critical during Proteus infection. Since Pta is surface associated and important for the persistence of P. mirabilis in the host, it was selected as a vaccine candidate. Mice intranasally vaccinated with a site-directed (indicated by an asterisk) (S366A) mutant purified intact toxin (Pta*) or the passenger domain Pta-α*, each independently conjugated with cholera toxin (CT), had significantly lower bacterial counts in their kidneys ( P = 0.001) and spleens (P = 0.002) than mice that received CT alone. The serum immunoglobulin G levels correlated with protection (P = 0.03). This is the first report describing the in vivo cytotoxicity and antigenicity of an autotransporter in P. mirabilis and its use in vaccine development.

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