Comparative proteomic analysis of Neisseria meningitidis wildtype and dprA null mutant strains links DNA processing to pilus biogenesis

Abstract

Background: DNA processing chain A (DprA) is a DNA binding protein which is ubiquitous in bacteria, and is required for DNA transformation to various extents among bacterial species. However, the interaction of DprA with competence and recombination proteins is poorly understood. Therefore, the proteomes of whole Neisseria meningitidis (Nm) wildtype and dprA mutant cells were compared. Such a comparative proteomic analysis increases our understanding of the interactions of DprA with other Nm components and may elucidate its potential role beyond DNA processing in transformation. Results: Using label-free quantitative proteomics, a total of 1057 unique Nm proteins were identified, out of which 100 were quantified as differentially abundant (P ≤ 0.05 and fold change ≥ |2|) in the dprA null mutant. Proteins involved in homologous recombination (RecA, UvrD and HolA), pilus biogenesis (PilG, PilT1, PilT2, PilM, PilO, PilQ, PilF and PilE), cell division, including core energy metabolism, and response to oxidative stress were downregulated in the Nm dprA null mutant. The mass spectrometry data are available via ProteomeXchange with identifier PXD006121. Immunoblotting and co-immunoprecipitation were employed to validate the association of DprA with PilG. The analysis revealed reduced amounts of PilG in the dprA null mutant and reduced amounts of DprA in the Nm pilG null mutant. Moreover, a number of pilus biogenesis proteins were shown to interact with DprA and /or PilG. Conclusions: DprA interacts with proteins essential for Nm DNA recombination in transformation, pilus biogenesis, and other functions associated with the inner membrane. Inverse downregulation of Nm DprA and PilG expression in the corresponding mutants indicates a link between DNA processing and pilus biogenesis.

Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis

“…PilG exhibits intrinsic DUS-independent DNA-binding activity and interacts with the PilQ secretin in the outer membrane [70]. Recently, a link between pilus biogenesis and processing of incoming DNA was proposed based on the observation that knockout of pilG and dprA , which loads RecA onto single-stranded DNA, resulted in reciprocal downregulation of the corresponding proteins [71]. TfpC, which was only recently identified as a new type IV pilus biogenesis factor, is located in the periplasm and harbours an N-terminal transmembrane domain anchoring the protein to the inner membrane.…”

Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059

“…The methods of proteomic analyses were modified from Beyene et al [49]. Briefly, all the statistical analyses were performed using Perseus software (version 1.6.14.0, Max-Planck Institute of Biochemistry, Martinsried, Germany), https://maxquant.net/perseus/ (accessed 20-09-2020)), and all results were filtered to a 1% false discovery rate (FDR).…”

“…The normalized abundance values for each protein were log2 transformed, and at least two valid values were required in the bio-triplicates for quantitation. When the original signals were zero, they were imputed with random numbers from a normal distribution, in which the mean and standard deviation were chosen from low abundance values below the noise level (Width = 0.3; shift = 1.8) [49,50]. To identify proteins with significantly different abundances when S4059 were grown on chitin compared to on mannose, the FDR were estimated using Benjamini-Hochberg method, and a two-tailed unpaired t-test was used with a combination of p-value ≤ 0.05 and Log2 fold-change ≥1.5 [51].…”

Factor H binding protein (fHbp)-mediated differential complement resistance of a serogroup C Neisseria meningitidis isolate from cerebrospinal fluid of a patient with invasive meningococcal disease

“…Proteome comparison techniques have been previously applied to N. meningitidis isolates to measure protein expression under different culture conditions as well as to investigate protein links and functions [16; 17; 18]. We then investigated differences in the phenotypes of the isolates by measuring their ability to survive in human serum and investigated underlying genetic mechanisms to better understand how the observed evolutionary changes may have occurred.…”

“…In the present study, we employed comparative proteomics to further characterize the Case and Carrier isolates from the 1997 University of Southampton outbreak to identify proteins differentially expressed that could have affected the ability of the Case isolate to cause disease and death of a student. Proteome comparison techniques have been previously applied to N. meningitidis isolates to measure protein expression under different culture conditions as well as to investigate protein links and functions [ 16 ; 17 ; 18 ]. We then investigated differences in the phenotypes of the isolates by measuring their ability to survive in human serum and investigated underlying genetic mechanisms to better understand how the observed evolutionary changes may have occurred.…”