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A combination of genomics and transcriptomics provides insights into the distribution and differential mRNA expression of type VI secretion system in clinical Klebsiella pneumoniae.

ABSTRACT: The type VI secretion system (T6SS) serves as a crucial molecular weapon in interbacterial competition and significantly influences the adaptability of bacteria in their ecological niche. However, the distribution and function of T6SS in clinical Klebsiella pneumoniae, a common opportunistic nosocomial pathogen, have not been fully elucidated. Here, we conducted a genomic analysis of 65 clinical K. pneumoniae isolates obtained from patients with varying infections. Genes encoding a T6SS cluster present in all analyzed strains of K. pneumoniae, and strains with identical sequence type carried structurally and numerically identical T6SS. Our study also highlights the importance of selecting conserved regions within essential T6SS genes for PCR-based identification of T6SS in bacteria. Afterward, we utilized the predominant sequence type 11 (ST11) K. pneumoniae HS11286 to investigate the effect of knocking out T6SS marker genes hcp or vgrG. Transcriptome analysis identified a total of 1,298 co-upregulated and 1,752 co-downregulated differentially expressed genes in both mutants. Pathway analysis showed that only Δhcp mutant exhibited alterations in transport, establishment of localization, localization, and cell processes. The absence of hcp or vgrG gene suppressed the expression of other T6SS-related genes within the locus I cluster. Additionally, interbacterial competition experiments showed that hcp and vgrG are essential for competitive ability of ST11 K. pneumoniae HS11286. This study furthers our understanding of the genomic characteristics of T6SS in clinical K. pneumoniae and suggests the involvement of multiple genes in T6SS of strain HS11286.

Importance

Gram-negative bacteria use type VI secretion system (T6SS) to deliver effectors that interact with neighboring cells for niche advantage. Klebsiella pneumoniae is an opportunistic nosocomial pathogen that often carries multiple T6SS loci, the function of which has not yet been elucidated. We performed a genomic analysis of 65 clinical K. pneumoniae strains isolated from various sources, confirming that all strains contained T6SS. We then used transcriptomics to further study changes in gene expression and its effect on interbacterial competition following the knockout of key T6SS genes in sequence type 11 (ST11) K. pneumoniae HS11286. Our findings revealed the distribution and genomic characteristics of T6SS in clinical K. pneumoniae. This study also described the overall transcriptional changes in the predominant Chinese ST11 strain HS11286 upon deletion of crucial T6SS genes. Additionally, this work provides a reference for future research on the identification of T6SS in bacteria.

SUBMITTER: Li W

PROVIDER: S-EPMC10964426 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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A combination of genomics and transcriptomics provides insights into the distribution and differential mRNA expression of type VI secretion system in clinical Klebsiella pneumoniae.

Li Wanzhen W Huang Xiaolan X Li Dan D Liu Xiaofen X Jiang Xiaoying X Bian Xingchen X Li Xin X Zhang Jing J

mSphere 20240304 3

The type VI secretion system (T6SS) serves as a crucial molecular weapon in interbacterial competition and significantly influences the adaptability of bacteria in their ecological niche. However, the distribution and function of T6SS in clinical Klebsiella pneumoniae, a common opportunistic nosocomial pathogen, have not been fully elucidated. Here, we conducted a genomic analysis of 65 clinical K. pneumoniae isolates obtained from patients with varying infections. Genes encoding a ...[more]

PMID: 38436228

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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:Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possesses an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition and identified VgrG4 as a T6SS effector. The DUF2345 domain of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.

Project description:Klebsiella pneumoniae is a nosocomial opportunistic pathogen that can cause pneumonia, liver abscesses, and infections of the bloodstream. The resistance and pathogenicity of K. pneumoniae pose major challenges to clinical practice. However, the ecology and pathogenic mechanisms of K. pneumoniae have not been fully elucidated. Among these mechanisms, the secretion systems encoded by strains of the bacteria confer adaptive advantages depending on the niche occupied. The type VI secretion system (T6SS) is a multi-protein complex that delivers effector proteins to the extracellular environment or directly to eukaryotic or prokaryotic cells. T6SSs are widely distributed in Gram-negative bacteria and play an important role in bacterial virulence and the interactions between bacteria and other microorganisms or the environment. This study aimed to enhance the understanding of the characteristics of T6SSs in K. pneumoniae through an in-depth comparative genomic analysis of the T6SS in 241 sequenced strains of K. pneumoniae. We identified the T6SS loci, the synteny of the loci in different species, as well as the effectors and core T6SS-related genes in K. pneumoniae. The presence of a T6SS was a common occurrence in K. pneumoniae, and two T6SS clusters are the most prevalent. The variable region downstream of the gene vgrG usually encodes effector proteins. Conserved domain analysis indicated that the identified putative effectors in K. pneumoniae had the functions of lipase, ribonuclease, deoxyribonuclease, and polysaccharide hydrolase. However, some effectors did not contain predicted functional domains, and their specific functions have yet to be elucidated. This in silico study represents a detailed analysis of T6SS-associated genes in K. pneumoniae and provides a foundation for future studies on the mechanism(s) of T6SSs, especially effectors, which may generate new insights into pathogenicity and lead to the identification of proteins with novel antimicrobial properties.

Project description:PurposeThe type VI system (T6SS) has the potential to be a new virulence factor for hypervirulent Klebsiella pneumoniae (hvKp) strains. This study aimed to characterize the molecular and clinical features of T6SS-positive and T6SS-negative K. pneumoniae isolates that cause abscesses.Patients and methodsA total of 169 non-duplicate K. pneumoniae strains were isolated from patients with abscesses in a tertiary hospital in China from January 2018 to June 2022, and clinical data were collected. For all isolates, capsular serotypes, T6SS genes, virulence, and drug resistance genes, antimicrobial susceptibility testing, and biofilm formation assays were assessed. Multilocus sequence typing was used to analyze the genotypes of hvKp. T6SS-positive hvKp, T6SS-negative hvKp, T6SS-positive cKP, and T6SS-negative cKP (n = 4 strains for each group) were chosen for the in vivo Galleria mellonella infection model and in vitro competition experiments to further explore the microbiological characteristics of T6SS-positive K. pneumoniae isolates.ResultsThe positive detection rate for T6SS was 36.1%. The rates of hvKp, seven virulence genes, K1 capsular serotype, and ST23 in T6SS-positive strains were all higher than those in T6SS-negative strains (p < 0.05). Multivariate logistic regression analysis indicated that the carriage of aerobactin (OR 0.01) and wcaG (OR 33.53) were independent risk factors for T6SS-positive strains (p < 0.05). The T6SS-positive strains had a stronger biofilm-forming ability than T6SS-negative strains (p < 0.05). The T6SS-positive and T6SS-negative strains showed no significant differences in competitive ability (p = 0.06). In the in vivo G. mellonella infection model, the T6SS(+)/hvKP group had the worst prognosis. Except for cefazolin and tegacyclin, T6SS-positive isolates displayed a lower rate of antimicrobial resistance to other drugs (p < 0.05). The T6SS-positive isolates were more likely to be acquired from community infections (p < 0.05).ConclusionKlebsiella pneumoniae isolates causing abscesses have a high prevalence of T6SS genes. T6SS-positive K. pneumoniae isolates are associated with virulence, and the T6SS genes may be involved in the hvKp virulence mechanism.

Project description:Silicate mineral weathering (dissolution) plays important roles in soil formation and global biogeochemical cycling. In this study, a combination of genomics, transcriptomics, and genetics was used to identify the molecular basis of mineral weathering activity and acid tolerance in Pseudomonas azotoformans F77. Biotite was chosen as a silicate mineral to investigate mineral weathering. The genome of strain F77 was sequenced, and the genes significantly upregulated when grown in the presence of biotite included mineral weathering-related genes associated with gluconic acid metabolism, flagellar assembly, and pilus biosynthesis and acid tolerance-related genes associated with neutralizing component production, reducing power, and proton efflux. The biotite-weathering behaviors of strain F77 and its mutants that were created by deleting the tkt, tal, and gntP genes, which are involved in gluconic acid metabolism, and the potF, nuoF, and gdtO genes, which are involved in acid tolerance, were determined. The Fe and Al concentrations in the strain F77-inoculated medium increased 2.2- to 13.7-fold compared to the controls. The cell numbers of strain F77 increased over time, while the pH values in the medium ranged from 3.75 to 3.90 between 20 and 36 h of incubation. The release of Al and Fe was significantly reduced in the F77 Δtal, F77 ΔgntP, F77 ΔpotF, and F77 ΔnuoF mutants. Bacterial growth was significantly reduced in the presence of biotite in the F77 ΔpotF and F77 ΔnuoF mutants. Our results demonstrated the acid tolerance of strain F77 and suggested that multiple genes and metabolic pathways in strain F77 are involved in biotite weathering and acid tolerance during the mineral weathering process. IMPORTANCE Acid production and tolerance play important roles in effective and persistent mineral weathering in bacteria, although the molecular mechanisms governing acid production and acid tolerance in bacteria have not been fully elucidated. In this study, the molecular mechanisms underlying biotite (as a silicate mineral) weathering (dissolution) and acid tolerance of P. azotoformans F77 were characterized using genomics, transcriptomics, and genetics analyses. Our results showed that the genes and metabolic pathways for gluconic acid metabolism, flagellar assembly, and pilus biosynthesis may play important roles in mineral weathering by strain F77. Notably, the genes associated with neutralizing component production, reducing power, and proton efflux may be related to acid tolerance in strain F77. The expression of these acid production- and acid tolerance-related genes was observed to be increased by biotite in strain F77. Our findings may help to elucidate the molecular mechanisms governing mineral weathering and, especially, acid tolerance in mineral-weathering bacteria.

Project description:ObjectiveTo analyze the distribution of blaOXA among global Klebsiella pneumoniae and the characteristics of blaOXA-carrying K. pneumoniae.Materials and methodsThe genomes of global K. pneumoniae were downloaded from NCBI by Aspera software. After quality check, the distribution of blaOXA among the qualified genomes was investigated by annotation with the resistant determinant database. The phylogenetic tree was constructed for the blaOXA variants based on the single nucleotide polymorphism (SNP) to explore the evolutionary relationship between these variants. The MLST (multi-locus sequence type) website and blastn tools were utilized to determine the sequence types (STs) of these blaOXA-carrying strains. and sample resource, isolation country, date and host were extracted by perl program for analyzing the characteristics of these strains.ResultsA total of 12,356 K. pneumoniae genomes were downloaded and 11,429 ones were qualified. Among them, 4386 strains were found to carry 5610 blaOXA variants which belonged to 27 varieties of blaOXAs, blaOXA-1 (n = 2891, 51.5%) and blaOXA-9 (n = 969, 17.3%) were the most prevalent blaOXA variants, followed by blaOXA-48 (n = 800, 14.3%) and blaOXA-232 (n = 480, 8.6%). The phylogenetic tree displayed 8 clades, three of them were composed of carbapenem-hydrolyzing oxacillinase (CHO). Totally, 300 distinct STs were identified among 4386 strains with ST11 (n = 477, 10.9%) being the most predominant one followed by ST258 (n = 410, 9.4%). Homo sapiens (2696/4386, 61.5%) was the main host for blaOXA-carrying K. pneumoniae isolates. The blaOXA-9-carrying K. pneumoniae strains were mostly found in the United States and blaOXA-48-carrying K. pneumoniae strains were mainly distributed in Europe and Asia.ConclusionAmong the global K. pneumoniae, numerous blaOXA variants were identified with blaOXA-1, blaOXA-9, blaOXA-48 and blaOXA-232 being the most prevalent ones, indicating that blaOXA rapidly evolved under the selective pressure of antimicrobial agents. ST11 and ST258 were the main clones for blaOXA-carrying K. pneumoniae.

Dataset Information

A combination of genomics and transcriptomics provides insights into the distribution and differential mRNA expression of type VI secretion system in clinical Klebsiella pneumoniae.

Importance

Publications

A combination of genomics and transcriptomics provides insights into the distribution and differential mRNA expression of type VI secretion system in clinical <i>Klebsiella pneumoniae</i>.

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