Copper(II)-Bis(Thiosemicarbazonato) Complexes as Antibacterial Agents: Insights into Their Mode of Action and Potential as Therapeutics.

There is increasing interest in the use of lipophilic copper (Cu)-containing complexes to combat bacterial infections. In this work, we showed that Cu complexes with bis(thiosemicarbazone) ligands [Cu(btsc)] exert antibacterial activity against a range of medically significant pathogens. Previous work using Neisseria gonorrhoeae showed that Cu(btsc) complexes may act as inhibitors of respiratory dehydrogenases in the electron transport chain. We now show that these complexes are also toxic against pathogens that lack a respiratory chain. Respiration in Escherichia coli was slightly affected by Cu(btsc) complexes, but our results indicate that, in this model bacterium, the complexes act primarily as agents that deliver toxic Cu ions efficiently into the cytoplasm. Although the chemistry of Cu(btsc) complexes may dictate their mechanism of action, their efficacy depends heavily on bacterial physiology. This is linked to the ability of the target bacterium to tolerate Cu and, additionally, the susceptibility of the respiratory chain to direct inhibition by Cu(btsc) complexes. The physiology of N. gonorrhoeae, including multidrug-resistant strains, makes it highly susceptible to damage by Cu ions and Cu(btsc) complexes, highlighting the potential of Cu(btsc) complexes (and Cu-based therapeutics) as a promising treatment against this important bacterial pathogen.

Overproduction of the MtrCDE efflux pump in Neisseria gonorrhoeae produces unexpected changes in cellular transcription patterns.

The global consequence of drug efflux gene overexpression in bacteria has not been specifically analyzed because strains showing high-level expression typically have mutations in genes encoding regulatory proteins that control other genes. Results from a transcriptional profiling study performed with a strain of Neisseria gonorrhoeae that is capable of high-level transcription of the mtrCDE efflux pump operon independently of control by cognate regulatory proteins revealed that its overexpression has ramifications for systems other than drug efflux.

Canary in the Coal Mine: How Resistance Surveillance in Commensals Could Help Curb the Spread of AMR in Pathogenic Neisseria.

Antimicrobial resistance (AMR) is widespread within Neisseria gonorrhoeae populations. Recent work has highlighted the importance of commensal Neisseria (cN) as a source of AMR for their pathogenic relatives through horizontal gene transfer (HGT) of AMR alleles, such as mosaic penicillin binding protein 2 (penA), multiple transferable efflux pump (mtr), and DNA gyrase subunit A (gyrA) which impact beta-lactam, azithromycin, and ciprofloxacin susceptibility, respectively. However, nonpathogenic commensal species are rarely characterized. Here, we propose that surveillance of the universally carried commensal Neisseria may play the role of the "canary in the coal mine," and reveal circulating known and novel antimicrobial resistance determinants transferable to pathogenic Neisseria. We summarize the current understanding of commensal Neisseria as an AMR reservoir, and call to increase research on commensal Neisseria species, through expanding established gonococcal surveillance programs to include the collection, isolation, antimicrobial resistance phenotyping, and whole-genome sequencing (WGS) of commensal isolates. This will help combat AMR in the pathogenic Neisseria by: (i) determining the contemporary AMR profile of commensal Neisseria, (ii) correlating AMR phenotypes with known and novel genetic determinants, (iii) qualifying and quantifying horizontal gene transfer (HGT) for AMR determinants, and (iv) expanding commensal Neisseria genomic databases, perhaps leading to the identification of new drug and vaccine targets. The proposed modification to established Neisseria collection protocols could transform our ability to address AMR N. gonorrhoeae, while requiring minor modifications to current surveillance practices. IMPORTANCE Contemporary increases in the prevalence of antimicrobial resistance (AMR) in Neisseria gonorrhoeae populations is a direct threat to global public health and the effective treatment of gonorrhea. Substantial effort and financial support are being spent on identifying resistance mechanisms circulating within the gonococcal population. However, these surveys often overlook a known source of resistance for gonococci-the commensal Neisseria. Commensal Neisseria and pathogenic Neisseria frequently share DNA through horizontal gene transfer, which has played a large role in rendering antibiotic therapies ineffective in pathogenic Neisseria populations. Here, we propose the expansion of established gonococcal surveillance programs to integrate a collection, AMR profiling, and genomic sequencing pipeline for commensal species. This proposed expansion will enhance the field's ability to identify resistance in and from nonpathogenic reservoirs and anticipate AMR trends in pathogenic Neisseria.

Antimicrobial Resistance Profiles of Human Commensal Neisseria Species.

Pathogenic Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea. N. gonorrhoeae has evolved high levels of antimicrobial resistance (AR) leading to therapeutic failures even in dual-therapy treatment with azithromycin and ceftriaxone. AR mechanisms can be acquired by genetic transfer from closely related species, such as naturally competent commensal Neisseria species. At present, little is known about the antimicrobial resistance profiles of commensal Neisseria. Here, we characterized the phenotypic resistance profile of four commensal Neisseria species (N. lactamica, N. cinerea, N. mucosa, and N. elongata) against 10 commonly used antibiotics, and compared their profiles to 4 N. gonorrhoeae strains, using disk diffusion and minimal inhibitory concentration assays. Overall, we observed that 3 of the 4 commensals were more resistant to several antibiotics than pathogenic N. gonorrhoeae strains. Next, we compared publicly available protein sequences of known AR genes, including penicillin-binding-protein 2 (PBP2) from commensals and N. gonorrhoeae strains. We found mutations in PBP2 known to confer resistance in N. gonorrhoeae also present in commensal Neisseria sequences. Our results suggest that commensal Neisseria have unexplored antibiotic resistance gene pools that may be exchanged with pathogenic N. gonorrhoeae, possibly impairing drug development and clinical treatment.

Polyamines can increase resistance of Neisseria gonorrhoeae to mediators of the innate human host defense.

Polyamines are biogenic polycationic molecules involved in key cellular functions. Extracellular polyamines found in bodily fluids or laboratory media can be imported by bacteria or bind to negatively charged bacterial surface structures, where they can impair binding of antimicrobials. We hypothesized that the presence of polyamines in fluids that bathe urogenital mucosal surfaces could alter the susceptibility of the sexually transmitted strict human pathogen Neisseria gonorrhoeae to mediators of the innate host defense. Herein we report that polyamines can significantly increase gonococcal resistance to two structurally diverse cationic antimicrobial peptides (polymyxin B and LL-37) but not to antibiotics that exert activity in the cytosol or periplasm (e.g., ciprofloxacin, spectinomycin, or penicillin). The capacity of polyamines to increase gonococcal resistance to cationic antimicrobial peptides was dose dependent, correlated with the degree of cationicity, independent of a polyamine transport system involving the polyamine permeases PotH and PotI, and was reversible. In addition, we found that polyamines increase gonococcal resistance to complement-mediated killing by normal human serum. We propose that polyamines in genital mucosal fluids may enhance gonococcal survival during infection by reducing bacterial susceptibility to host-derived antimicrobials that function in innate host defense.

Commensal Neisseria Kill Neisseria gonorrhoeae through a DNA-Dependent Mechanism.

The mucosa is colonized with commensal Neisseria. Some of these niches are sites of infection for the STD pathogen Neisseria gonorrhoeae (Ngo). Given the antagonistic behavior of commensal bacteria toward their pathogenic relatives, we hypothesized that commensal Neisseria may negatively affect Ngo colonization. Here, we report that commensal species of Neisseria kill Ngo through a mechanism based on genetic competence and DNA methylation state. Specifically, commensal-triggered killing occurs when the pathogen takes up commensal DNA containing a methylation pattern that it does not recognize. Indeed, any DNA will kill Ngo if it can enter the cell, is differentially methylated, and has homology to the pathogen genome. Consistent with these findings, commensal Neisseria elongata accelerates Ngo clearance from the mouse in a DNA-uptake-dependent manner. Collectively, we propose that commensal Neisseria antagonizes Ngo infection through a DNA-mediated mechanism and that DNA is a potential microbicide against this highly drug-resistant pathogen.

Characterization of a spermine/spermidine transport system reveals a novel DNA sequence duplication in Neisseria gonorrhoeae.

During infection, Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, comes into contact with numerous host compounds including polyamines (e.g. spermine and spermidine). Here, we show that spermine and spermidine concentrations in the growth medium decrease to undetectable levels in the presence of gonococci over time, but not when proteins of the putative polyamine transport system are lost due to mutation. We propose that gonococci have a functional and sole polyamine transport system (PotFGHI) that specifically imports spermine and spermidine. Bioinformatics and molecular analyses showed that the transporter's potGHI genes are organized as an operon while the gene encoding the necessary cognate periplasmic polyamine-binding protein (PotF) is located elsewhere on the chromosome. Interestingly, within the potGHI locus, we identified a novel duplicated sequence, which we term the Pot-Gene-Associated-Duplication-Element, present in variable copy numbers in different gonococcal strains that was likely formed from the 5(') and 3(') ends of the coding sequences of the tandemly linked potH and potG genes, respectively.

Spermine impairs biofilm formation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted infection termed gonorrhea. Recent reports indicate that gonococci can form a biofilm in vivo and under laboratory conditions. It is unclear, however, if formation of such biofilms or their dispersal are influenced by host factors that would be encountered during infection. In this respect, physiological levels of polyamines have been reported to influence biofilm structures formed by other Gram-negative bacteria as well those formed by Gram-positive bacteria and can cause dispersal of a biofilm formed by Bacillus subtilis. Based on these reports, we examined the influence of polyamines on gonococcal biofilm formation and their dispersal. We now report that physiological levels of certain polyamines, notably spermine, can significantly decrease the capacity of gonococci to form a biofilm, but do not cause dispersal of a preformed biofilm. In the context of natural gonococcal infection, the presence of physiological levels of spermine may be antagonistic for gonococci to form a biofilm and this may be of importance in the spread of the pathogen from a localized region.

Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens.

The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for 'proline racemase' virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not 'one way' directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on constraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host.

Crystal structure, catalytic mechanism, and mitogenic properties of Trypanosoma cruzi proline racemase.

Amino acid racemases catalyze the stereoinversion of the chiral C alpha to produce the d-enantiomers that participate in biological processes, such as cell wall construction in prokaryotes. Within this large protein family, bacterial proline racemases have been extensively studied as a model of enzymes acting with a pyridoxal-phosphate-independent mechanism. Here we report the crystal structure of the proline racemase from the human parasite Trypanosoma cruzi (TcPRACA), a secreted enzyme that triggers host B cell polyclonal activation, which prevents specific humoral immune responses and is crucial for parasite evasion and fate. The enzyme is a homodimer, with each monomer folded in two symmetric alpha/beta subunits separated by a deep crevice. The structure of TcPRACA in complex with a transition-state analog, pyrrole-2-carboxylic acid, reveals the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid/base catalysis through a carbanion stabilization mechanism. Mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein. In contrast, inhibitor binding promotes the closure of the interdomain crevice and completely abrogates B cell proliferation, suggesting that the mitogenic properties of TcPRACA depend on the exposure of transient epitopes in the ligand-free enzyme.

Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity.

Polyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect-transmitted protozoan responsible for Chagas' disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B-cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcPRACA paralogue revealed putative signals allowing the generation of an additional, non-secreted isoform of proline racemase by an alternative trans-splicing mechanism. Here, we demonstrate that overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock-down parasites. These results strongly emphasize that TcPRAC is a potential target for drug design as well as for immunomodulation of parasite-induced B-cell polyclonal activation.