Biology subjects

Tomberg, J.

Publications and source records attributed to Tomberg, J..

3 recordsLinked to original sources

bioRxiv2025

Antibiotic-resistance mutations in penicillin-binding protein 2 from the ceftriaxone-resistant Neisseria gonorrhoeae strain H041 strike a delicate balance between increasing resistance and maintaining transpeptidase activity

The mosaic penA allele (penA41) from H041, the most ceftriaxone-resistant Neisseria gonorrhoeae strain identified to date, encodes a variant of the essential Penicillin-Binding Protein 2 (PBP2) with 60 amino acid mutations compared to PBP2 from the antimicrobial-susceptible strain, FA19. Based on previous work from our lab and others, we identified a minimal set of 10 mutations that, when introduced into the {beta}-lactam antibiotic-susceptible penA allele from FA19 (penA19), confers two-thirds of the ceftriaxone and cefixime resistance compared to the penA41 allele. Three mutations (A311V, I312M, and V316P) are in the 2 helix of PBP2 containing the catalytic serine (Ser310), two (F504L and N512Y) are in the 3-4 loop that is important in binding and acylation, and one (G545S) interacts with conserved amino acids in the active site. The seventh mutation, T483S, confers substantial resistance to ceftriaxone within the minimal mutant set but requires the presence of three epistatic mutations located on the other side of the protein that do not alter resistance on their own yet are necessary to retain essential transpeptidase activity. These epistatic mutations change the backbone dihedral angles at position-447, which may increase flexibility of the enzyme and help restore essential transpeptidation. Our results highlight the complex balance necessary for evolving cephalosporin resistance while also retaining sufficient transpeptidase function in PBP2. Author SummaryIn this study, we set out to understand how Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is able to resist the last remaining recommended antibiotic, ceftriaxone. Gonorrhea is a common sexually transmitted infection worldwide, and rising resistance threatens to make it untreatable. We focused on penicillin-binding protein 2 (PBP2), which is essential for the bacteriums survival and is the lethal target of ceftriaxone. By incorporating a subset of the 60 PBP2 mutations found in a highly resistant strain into PBP2 from an antibiotic-susceptible strain, we discovered that resistance evolved from a combination of mutations that work together to directly reduce the capacity of ceftriaxone to inactivate the protein and others that act as "supporting" mutations to keep the protein functional despite the presence of the resistance mutations. Our study highlights how N. gonorrhoeae successfully negotiates the delicate balance between resistance and function to escape the lethal action of antibiotics.

microbiology↗

bioRxiv2023

Mutational analysis of LtgC, a lytic transglycosylase required for cell separation in Neisseria gonorrhoeae

Lytic transglycosylases function to degrade peptidoglycan strands that comprise the bacterial cell wall. Degradation of peptidoglycan at the septum following cell division is necessary for cell separation, and a deletion of ltgC in Neisseria gonorrhoeae results in growth in clusters of around 6-20 cells rather than as normal diplococci or monococci. N. gonorrhoeae LtgC is a homolog of Escherichia coli MltA, and comparison of the two proteins shows that LtgC has an extra domain not found in MltA, referred to as domain 3. To better understand the function of LtgC, we characterized N. gonorrhoeae mutants with substitutions in amino acids predicted to be necessary for enzymatic activity or amino acids predicted to be on the surface of domain 3, and we characterized a mutant lacking domain 3. All the mutants showed defects in cell separation, and the bacteria failed to release peptidoglycan-derived disaccharides into the medium. Purified LtgC proteins with the amino acid substitutions had reduced peptidoglycan degradation activity. LtgC was found to bind AmiC in bacterial 2-hybrid assays, and domain 3 mutations reduced binding. In human blood, an ltgC mutant showed decreased survival, suggesting the cell wall defects in the mutant make the bacteria more sensitive to innate immune system components. ImportanceNeisseria gonorrhoeae uses a smaller set of proteins for peptidoglycan breakdown compared to Escherichia coli or other model systems. The peptidoglycan breakdown that occurs at the septum following cell division in N. gonorrhoeae requires three proteins, amidase AmiC, amidase activator NlpD, and lytic transglycosylase LtgC. LtgC has an unusual structure that includes a third domain not found in related proteins. Using mutants that lacked LtgC activity or had amino acid changes in the third domain, we found that the extra domain is involved in interaction of LtgC with AmiC and that it is required for LtgC function for cell separation. All of the ltgC mutants examined showed reduced survival in blood, indicating the importance of LtgC activity for infection.

microbiology↗

bioRxiv2022

Commensal Neisseria species share immune suppressive mechanisms with Neisseria gonorrhoeae

Neisseria gonorrhoeae is a highly adapted human sexually transmitted pathogen that can cause symptomatic infections associated with localized inflammation as well as asymptomatic and subclinical infections, particularly in females. Gonococcal infection in humans does not generate an effective immune response in most cases, which contributes to both transmission of the pathogen and reinfection after treatment. Neisseria gonorrhoeae is known to evade and suppress human immune responses through a variety of mechanisms. Commensal Neisseria species that are closely related to N. gonorrhoeae, such as N. cinerea, N. lactamica, N. elongata, and N. mucosa, rarely cause disease and instead asymptomatically colonize mucosal sites for prolonged periods of time without evoking clearing immunologic responses. We have shown previously that N. gonorrhoeae inhibits the capacity of antigen-pulsed dendritic cells to induce CD4+ T cell proliferation in vitro. Much of the suppressive effects of N. gonorrhoeae on dendritic cells can be recapitulated either by outer-membrane vesicles released from the bacteria or by purified PorB, the most abundant outer-membrane protein in Neisseria gonorrhoeae. We show here that three commensal Neisseria species, N. cinerea, N. lactamica and N. mucosa, show a comparable capacity to suppress dendritic cell-induced T cell proliferation through mechanisms similar to those demonstrated previously for N. gonorrhoeae, including inhibition by purified PorB. Our findings suggest that some immune-evasive properties of pathogenic N. gonorrhoeae are shared with commensal Neisseria species and may contribute to the ability of both pathogens and commensals to cause prolonged mucosal colonization in humans.

microbiology↗