Abstract
Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt) strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as “AckA-dependent”. Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies focusing on specific acetylation sites that may have relevance in gonococcal pathogenesis and metabolism.
Highlights
IntroductionThe etiologic agent of gonorrhea, is a growing public health concern
Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is a growing public health concern
In our initial efforts to determine if lysine acetylation was present in N. gonorrhoeae, whole cell lysates from 1291wt grown for 3, 6, and 24 h were examined by Western blot using an anti-acetyl-lysine antibody (Fig 2)
Summary
The etiologic agent of gonorrhea, is a growing public health concern. N. gonorrhoeae infection can occur in both men and women, but the mechanisms and development of the infections differ. The increased level of multi-drug resistant N. gonorrhoeae strains has become a cause for concern. The Centers for Disease Control (CDC) recently recommended a change from antibiotic treatment with oral or injectable cephalosporin to using only injectable therapy as a means to slow the spread of antibiotic resistance to this antibiotic [2, 11, 12]. The increased incidence of infection, combined with the increase in strains with multi-drug resistance, led the CDC to prioritize N. gonorrhoeae as an “urgent” public health threat that needed to be addressed aggressively [13]
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