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RIL-seq reveals extensive involvement of small RNAs in virulence and capsule regulation in hypervirulent Klebsiella pneumoniae.

ABSTRACT: Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create 'superbugs' that are challenging to treat. Understanding virulence regulation of hvKp is thus critical. Accumulating evidence suggest that posttranscriptional regulation by small RNAs (sRNAs) plays a role in bacterial virulence, but it has hardly been studied in K. pneumoniae. We applied RIL-seq to a prototypical clinical isolate of hvKp to unravel the Hfq-dependent RNA-RNA interaction (RRI) network. The RRI network is dominated by sRNAs, including predicted novel sRNAs, three of which we validated experimentally. We constructed a stringent subnetwork composed of RRIs that involve at least one hvKp virulence-associated gene and identified the capsule gene loci as a hub target where multiple sRNAs interact. We found that the sRNA OmrB suppressed both capsule production and hypermucoviscosity when overexpressed. Furthermore, OmrB base-pairs within kvrA coding region and partially suppresses translation of the capsule regulator KvrA. This agrees with current understanding of capsule as a major virulence and fitness factor. It emphasizes the intricate regulatory control of bacterial phenotypes by sRNAs, particularly of genes critical to bacterial physiology and virulence.

SUBMITTER: Goh KJ

PROVIDER: S-EPMC11347178 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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RIL-seq reveals extensive involvement of small RNAs in virulence and capsule regulation in hypervirulent Klebsiella pneumoniae.

Goh Kwok Jian KJ Altuvia Yael Y Argaman Liron L Raz Yair Y Bar Amir A Lithgow Trevor T Margalit Hanah H Gan Yunn-Hwen YH

Nucleic acids research 20240801 15

Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create 'superbugs' that are challenging to treat. Understanding virulence regulation of hvKp is thus critical. Accumulating evidence suggest that posttranscriptional regulation by small RNAs (sRNAs) plays a role in bacterial virulence, but it has h ...[more]

PMID: 38804271

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Project description:The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to their high mortality rates and propensity to cause severe community-acquired infections in otherwise healthy individuals. The ability of hvKP to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Therefore, understanding the molecular mechanisms underlying hvKP virulence and biofilm formation is crucial for developing new therapeutic strategies. Polyphosphate Kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null-mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a shotgun proteomic analysis of the PPK1 mutant and wild-type strain to gain further insight into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins (DEP) involved in capsule synthesis (Wzi - Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB -LpxC - PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hypervirulent K. pneumoniae. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.

Project description:BackgroundKlebsiella pneumoniae, as a global priority pathogen, is well known for its capability of acquiring mobile genetic elements that carry resistance and/or virulence genes. Its virulence plasmid, previously deemed nonconjugative and restricted within hypervirulent K. pneumoniae (hvKP), has disseminated into classic K. pneumoniae (cKP), particularly carbapenem-resistant K. pneumoniae (CRKP), which poses alarming challenges to public health. However, the mechanism underlying its transfer from hvKP to CRKP is unclear.MethodsA total of 28 sequence type (ST) 11 bloodstream infection-causing CRKP strains were collected from Ruijin Hospital in Shanghai, China, and used as recipients in conjugation assays. Transconjugants obtained from conjugation assays were confirmed by XbaI and S1 nuclease pulsed-field gel electrophoresis, PCR detection and/or whole-genome sequencing. The plasmid stability of the transconjugants was evaluated by serial culture. Genetically modified strains and constructed mimic virulence plasmids were employed to investigate the mechanisms underlying mobilization. The level of extracellular polysaccharides was measured by mucoviscosity assays and uronic acid quantification. An in silico analysis of 2608 plasmids derived from 814 completely sequenced K. pneumoniae strains available in GenBank was performed to investigate the distribution of putative helper plasmids and mobilizable virulence plasmids.ResultsA nonconjugative virulence plasmid was mobilized by the conjugative plasmid belonging to incompatibility group F (IncF) from the hvKP strain into ST11 CRKP strains under low extracellular polysaccharide-producing conditions or by employing intermediate E. coli strains. The virulence plasmid was mobilized via four modes: transfer alone, cotransfer with the conjugative IncF plasmid, hybrid plasmid formation due to two rounds of single-strand exchanges at specific 28-bp fusion sites or homologous recombination. According to the in silico analysis, 31.8% (242) of the putative helper plasmids and 98.8% (84/85) of the virulence plasmids carry the 28-bp fusion site. All virulence plasmids carry the origin of the transfer site.ConclusionsThe nonconjugative virulence plasmid in ST11 CRKP strains is putatively mobilized from hvKP or E. coli intermediates with the help of conjugative IncF plasmids. Our findings emphasize the importance of raising public awareness of the rapid dissemination of virulence plasmids and the consistent emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains.

Project description:The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host's ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.

Project description:BackgroundCarbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is gradually becoming the dominant nosocomial pathogens in the healthcare setting.MethodsA retrospective study was conducted on patients with CR-KP from July 2021 to May 2022 in a teaching hospital. We identified bacterial isolates, collected the clinical data, and performed antimicrobial susceptibility testing, hypermucoviscosity string test, antimicrobial and virulence-associated genotype, as well as multi-locus sequence typing. CR-hvKP was defined as the presence of some combination of rmpA and/or rmpA2 with iucA, iroB, or peg-344. SPSS was used for data analysis. Univariate logistic regression analyses were used for risk factor and all statistically significant variables were included in the multivariate model. Statistical significance was taken to be P < 0.05.ResultsA total of 69 non-duplicated CR-KP isolates were collected, 27 of which were CR-hvKP. Out of the 69 CR-KP strains under investigation, they were distributed across 14 distinct sequence types (STs), wherein ST11 exhibited the highest prevalence, constituting 65.2% (45/69) of the overall isolates. The principal carbapenemase genes identified encompassed blakpc-2, blaNDM-1, and blaOXA-48, with blakpc-2 prevailing as the predominant type, accounting for 73.9% (51/69). A total of 69 CR-KP strains showed high resistance to common clinical antibiotics, with the exception of ceftazidime/avibactam. The ST11 (P = 0.040), ST65 (P = 0.030) and blakpc-2 ST11 clones (P = 0.010) were found to be highly related to hvKp. Regarding the host, tracheal intubation (P = 0.008), intracranial infection (P = 0.020) and neutrophil count (P = 0.049) were significantly higher in the patients with CR-hvKP. Multivariate analysis showed tracheal intubation to be an independent risk factor for CR-hvKP infection (P = 0.030, OR = 4.131). According to the clinical data we collected, tracheal intubation was performed mainly in the elderly with severe underlying diseases, which implied that CR-hvKP has become prevalent among elderly patients with comorbidities.ConclusionsThe prevalence of CR-hvKP may be higher than expected in the healthcare setting. CR-hvKP is gradually becoming the dominant nosocomial pathogen, and its prevalence and treatment will be a major challenge. It is essential to enhance clinical awareness and management of CR-hvKP infection.

Dataset Information

RIL-seq reveals extensive involvement of small RNAs in virulence and capsule regulation in hypervirulent Klebsiella pneumoniae.

Publications

RIL-seq reveals extensive involvement of small RNAs in virulence and capsule regulation in hypervirulent Klebsiella pneumoniae.

Similar Datasets

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