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Functional Insights From KpfR, a New Transcriptional Regulator of Fimbrial Expression That Is Crucial for Klebsiella pneumoniae Pathogenicity.

ABSTRACT: Although originally known as an opportunistic pathogen, Klebsiella pneumoniae has been considered a worldwide health threat nowadays due to the emergence of hypervirulent and antibiotic-resistant strains capable of causing severe infections not only on immunocompromised patients but also on healthy individuals. Fimbriae is an essential virulence factor for K. pneumoniae, especially in urinary tract infections (UTIs), because it allows the pathogen to adhere and invade urothelial cells and to form biofilms on biotic and abiotic surfaces. The importance of fimbriae for K. pneumoniae pathogenicity is highlighted by the large number of fimbrial gene clusters on the bacterium genome, which requires a coordinated and finely adjusted system to control the synthesis of these structures. In this work, we describe KpfR as a new transcriptional repressor of fimbrial expression in K. pneumoniae and discuss its role in the bacterium pathogenicity. K. pneumoniae with disrupted kpfR gene exhibited a hyperfimbriated phenotype with enhanced biofilm formation and greater adhesion to and replication within epithelial host cells. Nonetheless, the mutant strain was attenuated for colonization of the bladder in a murine model of urinary tract infection. These results indicate that KpfR is an important transcriptional repressor that, by negatively controlling the expression of fimbriae, prevents K. pneumoniae from having a hyperfimbriated phenotype and from being recognized and eliminated by the host immune system.

SUBMITTER: Gomes AEI

PROVIDER: S-EPMC7861041 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Functional Insights From KpfR, a New Transcriptional Regulator of Fimbrial Expression That Is Crucial for Klebsiella pneumoniae Pathogenicity.

Gomes Ana Érika Inácio AÉI Pacheco Thaisy T Dos Santos Cristiane da Silva CDS Pereira José Aires JA Ribeiro Marcelo Lima ML Darrieux Michelle M Ferraz Lúcio Fábio Caldas LFC

Frontiers in microbiology 20210121

Although originally known as an opportunistic pathogen, Klebsiella pneumoniae has been considered a worldwide health threat nowadays due to the emergence of hypervirulent and antibiotic-resistant strains capable of causing severe infections not only on immunocompromised patients but also on healthy individuals. Fimbriae is an essential virulence factor for K. pneumoniae, especially in urinary tract infections (UTIs), because it allows the pathogen to adhere and invade urothelial ce ...[more]

PMID: 33552015

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Project description:BackgroundKlebsiella pneumoniae (KP) is an opportunistic pathogen that mainly causes respiratory and urinary tract infections. The frequent occurrence of simultaneously virulent and multiple drug-resistant isolates led WHO to include this species in the list of top priorities for research and development of therapeutic alternatives. The comprehensive knowledge of the molecular mechanisms underlying KP virulence may lead to the proposal of more efficient and specific drugs. One of its virulence factors is the Type VI Secretion System (T6SS), which contributes to bacterial competition, cell invasion and in vivo colonisation. Despite the few studies showing the involvement of T6SS in KP pathogenesis, little is known concerning the regulation of its expression. The understanding of regulatory mechanisms may give more clues about the function of the system and the possibilities of future interference in this process. This work aimed to standardise the annotation of T6SS genes in KP strains and identify mechanisms of their transcriptional regulation through computational predictions.ResultsWe analyzed the genomes of Kp52.145, HS11286 and NTUH-K2044 strains to perform a broad prediction and re-annotation of T6SS genes through similarity searches, comparative and linear discriminant analysis. 38 genes were found in Kp52.145, while 29 in HS11286 and 30 in NTUH-K2044. Genes coding for iron uptake systems are encoded in adjacencies of T6SS, suggesting that KP T6SS might also play a role in ion import. Some of the T6SS genes are comprised in syntenic regions. 17 sigma 70-dependent promoter regions were identified in Kp52.145, 12 in HS11286 and 12 in NTUH-K2044. Using VirtualFootprint algorithm, binding sites for 13 transcriptional regulators were found in Kp52.145 and 9 in HS11286 and 17 in NTUH-K2044. Six of them are common to the 3 strains: OxyR, H-NS, RcsAB, GcvA, Fis, and OmpR.ConclusionsThe data presented herein are derived from computational analysis. Although future experimental studies are required to confirm those predictions, they suggest that KP T6SS might be regulated in response to environmental signals that are indeed sensed by the bacteria inside the human host: temperature (H-NS), nutrition-limitation (GcvA and Fis), oxidative stress (OxyR) and osmolarity (RscAB and OmpR).

Project description:Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of Gene ontology (GO) analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. In this study the ΔaflR and overexpression (OE) strains based on the well-established double-crossover recombinational technique were constructed to investigate the integrative function of the aflR gene in A. flavus. The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. The aflatoxin B1 (AFB1) of the ΔaflR strain was 180 ng/mL and aflatoxin B2 (AFB2) was 2.95 ng/mL on YES medium for 5 days, which was 1/1,000 of that produced by the wild-type strain (WT). In addition, the ΔaflR strain produced relatively sparse conidia and a very small number of sclerotia. On the seventh day, the sclerotia yield on each plate of the WT and OE strains exceeded 1,000, while the sclerotial formation of the ΔaflR strain was not detected until 14 days. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly downregulated. Meanwhile, the ΔaflR strain compared with the WT strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT. The results demonstrated that the A. flavus aflR gene also played a positive role in the fungal growth and development in addition to aflatoxin biosynthesis. IMPORTANCE Past studies of the A. flavus aflR gene and its orthologues in related Aspergillus species were solely focused on their roles in secondary metabolism. In this study, we used the ΔaflR and OE strains to demonstrate the role of aflR in growth and development of A. flavus. For the first time, we confirmed that the ΔaflR strain also was defective in production of conidia and sclerotia, asexual propagules of A. flavus. Our transcriptomic analysis further showed that genes involved in spore germination, sclerotial development, aflatoxin biosynssssthesis, and carbohydrate metabolism exhibited significant differences in the ΔaflR strain compared with the WT strain. Our study indicates that AflR not only plays an important role in regulating aflatoxin synthesis but also in playing a positive role in the conidial formation and sclerotial development in A. flavus. This study reveals the critical and positive role of the aflR gene in fungal growth and development, and provides a theoretical basis for the genetic studies of other aspergilli.

Project description:RarA is an AraC-type regulator in Klebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both the acrAB and oqxAB efflux genes. Increased rarA expression has also been shown to be integral in the development of tigecycline resistance in the absence of ramA in K. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8?rarA/pACrarA-2 (rarA-expressing construct) and Ecl8?rarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences of K. pneumoniae MGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show that rarA overexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141, sdaCB, and leuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g., nuoA, narJ, and proWX) were found to be downregulated. Biolog phenotype analyses demonstrated that rarA overexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype of K. pneumoniae.

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Functional Insights From KpfR, a New Transcriptional Regulator of Fimbrial Expression That Is Crucial for Klebsiella pneumoniae Pathogenicity.

Publications

Functional Insights From KpfR, a New Transcriptional Regulator of Fimbrial Expression That Is Crucial for <i>Klebsiella pneumoniae</i> Pathogenicity.

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