Drug Targeting a Gonococcal Virulence Factor Exploits Host Antimicrobial Peptides in Clearance of Infection

(See the Major Article by Gulati et al on pages 1641–50.

While our attention is rightfully fixated on the ongoing, devastating worldwide Covid-19 pandemic, it is important to remember the global antibiotic resistance crisis that continues to challenge our ability to treat bacterial infections. This challenge demands the development of new and effective drugs as well as novel strategies to combat antibiotic-resistant infections. Classically, conventional antibiotic development work has targeted essential systems required for bacterial growth under laboratory conditions. More recently, however, investigators have considered bacterial virulence factors as targets for therapeutic development because their loss would decrease damage to the host and promote clearance by natural defense systems. Although an antivirulence drug development strategy has theoretical appeal, heretofore few studies have provided in-depth experimental evidence for its application. In the current issue of the Journal of Infectious Diseases, this potential is highlighted by Gulati et al [1], who targeted a gonococcal virulence factor known to be critical for bacterial resistance to innate host defenses and survival during infection. Indeed, their data support an antivirulence-targeting strategy for treatment of gonococcal infections.

With an estimated 87 million cases of infection per year, gonorrhea remains a major global public health concern [2]. In the absence of a vaccine, continued antibiotic efficacy is crucial for both cure and reducing the spread of gonococcal infections in the community. Unfortunately, the emergence and spread of Neisseria gonorrhoeae strains resistant to approved antibiotics threatens gonorrhea control. Given the potential devastating consequences of gonococcal infections on reproductive health (especially for women), the prospect of untreatable infections is worrisome [3]. Although efforts at vaccine and antibiotic development are in progress, additional strategies to assure future efficacy of clinical treatment of gonorrhea are needed. In this respect, chemotherapeutic targeting of a gonococcal virulence factor is an option warranting consideration.

The gonococcal virulence factor examined by Gulati et al is the lipooligosaccharide (LOS) that is often capped by sialic acid at the lacto-N-neotetraose located within the α-chain or the terminal galactose of the β-chain [4]. This capping is mediated by sialyltransferase (Lst) and is important for gonococcal resistance to complement and certain host-derived antimicrobial peptides (AMPs) in the cathelicidin family such as murine CRAMP or human LL37. Furthermore, LOS sialylation enhances biofilm formation as well as survival in the lower genital tract of experimentally infected female mice and the urethra of experimentally infected human male volunteers. In sum, chemically targeting LOS sialylation is a logical option in the new drug development arena.

Lst catalyzes the transfer of N-acetylneuraminic acid (Neu5Ac; a member of the sialic acid family) from host cytidine 5′-monophosphate (CMP)-Neu5Ac. While genetic inactivation of lst does not result in a growth defect in laboratory media, it does render gonococci susceptible to complement and CRAMP/LL37. Earlier work revealed that CMP-Neu5Ac analogs (CMP-nonulosonates [CMP-NulOs] such as CMP-Leg5, 7Ac₂, and CMP-Kdn) can be substrates for Lst and can be added in lieu of Neu5Ac to terminal galactose LOS residues. Interestingly, capping of LOS by these analogs failed to endow gonococci with complement or cathelicidin resistance. Relevant to their therapeutic potential, Gulati et al [1] further showed that administration of Neu5ac analogs enhances clearance of gonococci from experimentally infected female mice, including a strain (H041) that caused the first reported case of third-generation cephalosporin resistance [5]. Justifiably, the authors expected that the loss of complement resistance due to LOS capping by Neu5Ac analogs would be the reason for enhanced elimination of gonococci in infected mice. Surprisingly, this was not the case. Instead, the authors found that loss of cathelicidin resistance correlated with increased clearance of gonococci. This conclusion was validated by comparison of gonococcal survival in wild-type versus CRAMP-deficient mice treated or untreated with Neu5Ac analogs.

What are the molecular mechanisms by which sialylation of LOS protects gonococci from the lethal action of CRAMP (LL37), and how does loss of this modification enhance bacterial susceptibility to clearance during experimental infection? In the first case, evidence has been presented that gonococci with this LOS modification bind these cathelicidins more effectively than isogenic variants unable to perform the Neu5Ac capping reaction due to loss of Lst. Thus, one can speculate that Neu5Ac at the LOS termini acts as a sink for cathelicidins, preventing their movement to the bacterial outer membrane surface where they traverse the cell envelope and reach their inner membrane target. Logically, loss of this shield due to the presence of a Neu5Ac analog would enhance the number of molecules that ultimately localize to the inner membrane. A second possibility to explain the efficacy of Neu5Ac analogs in promoting clearance of gonococci during infection embraces the known immunomodulatory activities of cathelicidins that stimulate chemokine/cytokine production and phagocyte influx [6]. Perhaps CRAMP (LL37) bound to Neu5Ac is incapable of performing these immunoregulatory actions? Regardless of the mechanism, the elegant work of Gulati et al [1] emphasizes the importance of cathelicidins in innate host defense and validates therapeutic strategies that indirectly accentuate their antimicrobial activity (directly or indirectly). While development of resistance to any antimicrobial is a concern, the authors posited that resistance of gonococci to Neu5Ac analogs would be counterproductive, as it would likely negate the ability of Lst to transfer Neu5Ac to LOS, thereby rendering gonococci susceptible to cathelicidins.

The emergence of a limited number of gonococcal strains resistant to azithromycin and ceftriaxone/cefixime, which are used in many countries as front-line antibiotics, is worrisome. This problem is especially concerning for low-resource countries where the costs of using more expensive antibiotics would be prohibitive. Thus, Neu5Ac analogs may be inexpensive alternatives to classical antibiotics. As these analogs promote increased bacterial susceptibility to membrane-damaging cathelicidins, they might also act as adjuvant therapeutics by increasing cell envelope permeability to antibiotics that poorly penetrate the cell envelope or have low tissue availability.

Notes

Disclaimer. The contents of this article is the sole responsibility of the author and does not necessarily reflect the views, opinions, or policies of the National Institutes of Health or the US Department of Veterans Affairs, or the United States Government.

Financial support. This work was supported by the National Institutes of Health (grant numbers R37 AI021150-34 and R01 AI AI147609-02). The author is a recipient of a Senior Research Career Scientist Award from the Biomedical Laboratory Research and Development Service, United States Department of Veterans Affairs.

Potential conflicts of interest. The author certifies no potential conflicts of interest. The author has submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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