Deletion Mutants of Schmallenberg Virus Are Avirulent and Protect from Virus Challenge

ABSTRACTSince its emergence, Schmallenberg virus (SBV), a novel insect-transmitted orthobunyavirus which predominantly infects ruminants, has caused a large epidemic in European livestock. Newly developed inactivated vaccines are available, but highly efficacious and safe live vaccines are still not available. Here, the properties of novel recombinant SBV mutants lacking the nonstructural protein NSs (rSBVΔNSs) or NSm (rSBVΔNSm) or both of these proteins (rSBVΔNSs/ΔNSm) were testedin vitroandin vivoin type I interferon receptor knockout mice (IFNAR−/−) and in a vaccination/challenge trial in cattle. As for other bunyaviruses, both nonstructural proteins of SBV are not essential for viral growthin vitro. In interferon-defective BHK-21 cells, rSBVΔNSs and rSBVΔNSm replicated to levels comparable to that of the parental rSBV; the double mutant virus, however, showed a mild growth defect, resulting in lower final virus titers. Additionally, both mutants with an NSs deletion induced high levels of interferon and showed a marked growth defect in interferon-competent sheep SFT-R cells. Nevertheless, in IFNAR−/−mice, all mutants were virulent, with the highest mortality rate for rSBVΔNSs and a reduced virulence for the NSm-deleted virus. In cattle, SBV lacking NSm caused viremia and seroconversion comparable to those caused by the wild-type virus, while the NSs and the combined NSs/NSm deletion mutant induced no detectable virus replication or clinical disease after immunization. Furthermore, three out of four cattle immunized once with the NSs deletion mutant and all animals vaccinated with the virus lacking both nonstructural proteins were fully protected against a challenge infection. Therefore, the double deletion mutant will provide the basis for further developments of safe and efficacious modified live SBV vaccines which could be also a model for other viruses of the Simbu serogroup and related orthobunyaviruses.IMPORTANCESBV induces only mild clinical signs in adult ruminants but causes severe fetal malformation and, thereby, can have an important impact on animal welfare and production. As SBV is an insect-transmitted pathogen, vaccination will be one of the most important aspects of disease control. Here, mutant viruses lacking one or two proteins that essentially contribute to viral pathogenicity were tested as modified live vaccines in cattle. It could be demonstrated that a novel recombinant double deletion mutant is a safe and efficacious vaccine candidate. This is the first description of a putative modified live vaccine for the complete genusOrthobunyavirus, and in addition, such a vaccine type has never been tested in cattle for any virus of the entire familyBunyaviridae. Therefore, the described vaccine also represents the first model for a broad range of related viruses and is of high importance to the field.

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CcpA Is Important for Growth and Virulence of Enterococcus faecium

Infection and Immunity ◽

10.1128/iai.01911-14 ◽

2014 ◽

Vol 82 (9) ◽

pp. 3580-3587 ◽

Cited By ~ 15

Author(s):

Sudha R. Somarajan ◽

Jung H. Roh ◽

Kavindra V. Singh ◽

George M. Weinstock ◽

Barbara E. Murray

Keyword(s):

Enterococcus Faecium ◽

Deletion Mutant ◽

Growth Delay ◽

Growth Defect ◽

Single Mutation ◽

Competitive Growth ◽

Content Type ◽

Mixed Inoculum ◽

Slight Growth

ABSTRACTThe collagen adhesin Acm was the first virulence determinant reported to be important for the pathogenesis ofEnterococcus faeciumin a rat infective endocarditis model. We had previously reported that there was a slight growth delay associated withacmallelic replacement (cat) mutant strain TX6051 used in that study. Recently, we generated a nonpolar markerlessacmdeletion mutant and did not observe a delay in growth. We therefore performed comparative genome sequence analysis of wild-type strain TX82 and TX6051 and found a single mutation, a nonsense mutation in theccpAgene of TX6051. After correcting this mutation, the growth defect of TX6051 was abolished, implicating a role for CcpA in the growth ofE. faecium. To confirm this, we created accpAdeletion mutant of TX82, which also exhibited a slight delay in growth. Furthermore, theccpAdeletion mutant was attenuated (P= 0.0024) in a mixed-inoculum (TX82 plus TX82 ΔccpA) rat endocarditis model and also in anin vitrocompetitive growth assay; accpA-complemented strain showed neither reduced growth nor reduced virulence. We also found attenuation in the endocarditis model with the newacmdeletion mutant although not as great as that previously observed with TX6051 carrying theccpAmutation. Taken together, our data confirm the role of Acm in the pathogenesis of endocarditis. We also show that CcpA affects the growth ofE. faecium, that an intactccpAgene is important for full virulence, and that accpAmutation was partly responsible for the highly attenuated phenotype of TX6051.

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Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens

Biomedicines ◽

10.3390/biomedicines9060679 ◽

2021 ◽

Vol 9 (6) ◽

pp. 679

Author(s):

Benedict-Uy Fabia ◽

Joshua Bingwa ◽

Jiyeon Park ◽

Nguyen-Mihn Hieu ◽

Jung-Hoon Ahn

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Pseudomonas Fluorescens ◽

Abc Transporter ◽

Transforming Growth Factor Beta ◽

Deletion Mutant ◽

Transforming Growth Factor ◽

Gene Knockout ◽

Type I ◽

Double Deletion

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF.

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Bacterial Enzymes Catalyzing the Synthesis of 1,8-Dihydroxynaphthalene, a Key Precursor of Dihydroxynaphthalene Melanin, fromSorangium cellulosum

Applied and Environmental Microbiology ◽

10.1128/aem.00258-18 ◽

2018 ◽

Vol 84 (9) ◽

Cited By ~ 8

Author(s):

Yusuke Sone ◽

Shuto Nakamura ◽

Makoto Sasaki ◽

Fumihito Hasebe ◽

Seung-Young Kim ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Optical Purity ◽

Brown Pigment ◽

Type I ◽

Enzymatic System ◽

Type Iii Polyketide Synthase ◽

Content Type ◽

Type Iii ◽

Bacterial Origin

ABSTRACT1,8-Dihydroxynaphthalene (1,8-DHN) is a key intermediate in the biosynthesis of DHN melanin, which is specific to fungi. In this study, we characterized the enzymatic properties of the gene products of an operon consisting ofsoceCHS1,bdsA, andbdsBfrom the Gram-negative bacteriumSorangium cellulosum. Heterologous expression ofsoceCHS1,bdsA, andbdsBinStreptomyces coelicolorcaused secretion of a dark-brown pigment into the broth. High-performance liquid chromatography (HPLC) analysis of the broth revealed that the recombinant strain produced 1,8-DHN, indicating that the operon encoded a novel enzymatic system for the synthesis of 1,8-DHN. Simultaneous incubation of the recombinant SoceCHS1, BdsA, and BdsB with malonyl-coenzyme A (malonyl-CoA) and NADPH resulted in the synthesis of 1,8-DHN. SoceCHS1, a type III polyketide synthase (PKS), catalyzed the synthesis of 1,3,6,8-tetrahydroxynaphthalene (T4HN)in vitro. T4HN was in turn converted to 1,8-DHN by successive steps of reduction and dehydration, which were catalyzed by BdsA and BdsB. BdsA, which is a member of the aldo-keto reductase (AKR) superfamily, catalyzed the reduction of T4HN and 1,3,8-tetrahydroxynaphthalene (T3HN) to scytalone and vermelone, respectively. The stereoselectivity of T4HN reduction by BdsA occurred on thesi-face to give (R)-scytalone with more than 99% optical purity. BdsB, a SnoaL2-like protein, catalyzed the dehydration of scytalone and vermelone to T3HN and 1,8-DHN, respectively. The fungal pathway for the synthesis of 1,8-DHN is composed of a type I PKS, naphthol reductases of the short-chain dehydrogenase/reductase (SDR) superfamily, and scytalone dehydratase (SD). These findings demonstrated 1,8-DHN synthesis by novel enzymes of bacterial origin.IMPORTANCEAlthough the DHN biosynthetic pathway was thought to be specific to fungi, we discovered novel DHN synthesis enzymes of bacterial origin. The biosynthesis of bacterial DHN utilized a type III PKS for polyketide synthesis, an AKR superfamily for reduction, and a SnoaL2-like NTF2 superfamily for dehydration, whereas the biosynthesis of fungal DHN utilized a type I PKS, SDR superfamily enzyme, and SD-like NTF2 superfamily. Surprisingly, the enzyme systems comprising the pathway were significantly different from each other, suggesting independent, parallel evolution leading to the same biosynthesis. DHN melanin plays roles in host invasion and adaptation to stress in pathogenic fungi and is therefore important to study. However, it is unclear whether DHN biosynthesis occurs in bacteria. Importantly, we did find that bacterial DHN biosynthetic enzymes were conserved among pathogenic bacteria.

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How the Anaerobic Enteropathogen Clostridioides difficile Tolerates Low O2 Tensions

mBio ◽

10.1128/mbio.01559-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Nicolas Kint ◽

Carolina Alves Feliciano ◽

Maria C. Martins ◽

Claire Morvan ◽

Susana F. Fernandes ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Anaerobic Bacteria ◽

Growth Defect ◽

Strictly Anaerobic ◽

Low Oxygen ◽

Air Exposure ◽

Vegetative Cells ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2. O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile. This enteropathogen has developed efficient strategies to detoxify O2. In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile. These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

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SSP3 Is a Novel Plasmodium yoelii Sporozoite Surface Protein with a Role in Gliding Motility

Infection and Immunity ◽

10.1128/iai.01800-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4643-4653 ◽

Cited By ~ 18

Author(s):

Anke Harupa ◽

Brandon K. Sack ◽

Viswanathan Lakshmanan ◽

Nadia Arang ◽

Alyse N. Douglass ◽

...

Keyword(s):

Salivary Glands ◽

Biological Activities ◽

Surface Protein ◽

Plasmodium Yoelii ◽

Gliding Motility ◽

Surface Proteins ◽

Type I ◽

Content Type ◽

Mosquito Midgut

ABSTRACTPlasmodiumsporozoites develop within oocysts in the mosquito midgut wall and then migrate to the salivary glands. After transmission, they embark on a complex journey to the mammalian liver, where they infect hepatocytes. Proteins on the sporozoite surface likely mediate multiple steps of this journey, yet only a few sporozoite surface proteins have been described. Here, we characterize a novel, conserved sporozoite surface protein (SSP3) in the rodent malaria parasitePlasmodium yoelii. SSP3 is a putative type I transmembrane protein unique toPlasmodium. By using epitope tagging and SSP3-specific antibodies in conjunction with immunofluorescence microscopy, we showed that SSP3 is expressed in mosquito midgut oocyst sporozoites, exhibiting an intracellular localization. In sporozoites derived from the mosquito salivary glands, however, SSP3 localized predominantly to the sporozoite surface as determined by immunoelectron microscopy. However, the ectodomain of SSP3 appeared to be inaccessible to antibodies in nonpermeabilized salivary gland sporozoites. Antibody-induced shedding of the major surface protein circumsporozoite protein (CSP) exposed the SSP3 ectodomain to antibodies in some sporozoites. Targeted deletion ofSSP3adversely affectedin vitrosporozoite gliding motility, which, surprisingly, impacted neither their cell traversal capacity, host cell invasionin vitro, nor infectivityin vivo. Together, these data reveal a previously unappreciated complexity of thePlasmodiumsporozoite surface proteome and the roles of surface proteins in distinct biological activities of sporozoites.

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Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication

Journal of Virology ◽

10.1128/jvi.00563-18 ◽

2018 ◽

Vol 92 (15) ◽

Cited By ~ 3

Author(s):

Paula F. Zamora ◽

Liya Hu ◽

Jonathan J. Knowlton ◽

Roni M. Lahr ◽

Rodolfo A. Moreno ◽

...

Keyword(s):

Virus Replication ◽

Viral Genome ◽

Nonstructural Protein ◽

Dsrna Virus ◽

Genome Replication ◽

Nonstructural Proteins ◽

Content Type ◽

The Family ◽

Viral Genome Replication

ABSTRACTViral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, usingin vitroand cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases the viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication.IMPORTANCEFollowing infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the familyReoviridaeencode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different viruses in the familyReoviridaediverge in primary sequence, they are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Usingin vitroand cell culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the familyReoviridaeassort and replicate their genomes.

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Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00140-14 ◽

2014 ◽

Vol 58 (6) ◽

pp. 3312-3326 ◽

Cited By ~ 12

Author(s):

B. K. Kishore Reddy ◽

Sudhir Landge ◽

Sudha Ravishankar ◽

Vikas Patil ◽

Vikas Shinde ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Present Report ◽

Kinetic Studies ◽

Antimycobacterial Activity ◽

Type I ◽

Pantothenate Kinase ◽

Content Type ◽

Rate Limiting Step

ABSTRACTPantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by thecoaAgene is an essential pantothenate kinase inMycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-typeM. tuberculosisstrain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in aM. tuberculosisknockdown strain with reduced PanK expression levels. Additionally,in vitroandin vivosurvival kinetic studies performed with aM. tuberculosisPanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK inM. tuberculosis.

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Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01889-19 ◽

2020 ◽

Vol 64 (3) ◽

Cited By ~ 1

Author(s):

Jixu Li ◽

Huanping Guo ◽

Eloiza May Galon ◽

Yang Gao ◽

Seung-Hun Lee ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Drug Targets ◽

Protozoan Parasite ◽

Cell Types ◽

Lytic Cycle ◽

Type I ◽

Content Type ◽

Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

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Na+/H+Antiport Is Essential for Yersinia pestis Virulence

Infection and Immunity ◽

10.1128/iai.00071-13 ◽

2013 ◽

Vol 81 (9) ◽

pp. 3163-3172 ◽

Cited By ~ 28

Author(s):

Yusuke Minato ◽

Amit Ghosh ◽

Wyatt J. Faulkner ◽

Erin J. Lind ◽

Sara Schesser Bartra ◽

...

Keyword(s):

Yersinia Pestis ◽

Drug Target ◽

Deletion Mutant ◽

Ex Vivo ◽

Ion Homeostasis ◽

Content Type ◽

Double Deletion ◽

Derivative Strain

ABSTRACTNa+/H+antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na+/H+antiport inYersinia pestisvirulence and found thatY. pestisstrains lacking the major Na+/H+antiporters, NhaA and NhaB, are completely attenuated in anin vivomodel of plague. TheY. pestisderivative strain lacking thenhaAandnhaBgenes showed markedly decreased survival in blood and blood serumex vivo. Complementation of eithernhaAornhaBintransrestored the survival of theY. pestis nhaA nhaBdouble deletion mutant in blood. ThenhaA nhaBdouble deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na+levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na+/H+antiport is indispensable for the survival ofY. pestisin the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused byY. pestisand possibly for those caused by other blood-borne bacterial pathogens.

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Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli

Infection and Immunity ◽

10.1128/iai.00369-16 ◽

2016 ◽

Vol 84 (11) ◽

pp. 3220-3231 ◽

Cited By ~ 9

Author(s):

Kumiko Kurabayashi ◽

Tomohiro Agata ◽

Hirofumi Asano ◽

Haruyoshi Tomita ◽

Hidetada Hirakawa

Keyword(s):

Escherichia Coli ◽

Epithelial Cells ◽

Antimicrobial Agents ◽

Host Cells ◽

Type I ◽

Wild Type ◽

Content Type ◽

Type I Fimbriae ◽

Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD , which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs.

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