Neisseria gonorrhoeae MlaA influences gonococcal virulence and membrane vesicle production

The six-component maintenance of lipid asymmetry (Mla) system is responsible for retrograde transport of phospholipids, ensuring the barrier function of the Gram-negative cell envelope. Located within the outer membrane, MlaA (VacJ) acts as a channel to shuttle phospholipids from the outer leaflet. We identified Neisseria gonorrhoeae MlaA (ngo2121) during high-throughput proteomic mining for potential therapeutic targets against this medically important human pathogen. Our follow-up phenotypic microarrays revealed that lack of MlaA results in a complex sensitivity phenome. Herein we focused on MlaA function in cell envelope biogenesis and pathogenesis. We demonstrate the existence of two MlaA classes among 21 bacterial species, characterized by the presence or lack of a lipoprotein signal peptide. Purified truncated N. gonorrhoeae MlaA elicited antibodies that cross-reacted with a panel of different Neisseria. Little is known about MlaA expression; we provide the first evidence that MlaA levels increase in stationary phase and under anaerobiosis but decrease during iron starvation. Lack of MlaA resulted in higher cell counts during conditions mimicking different host niches; however, it also significantly decreased colony size. Antimicrobial peptides such as polymyxin B exacerbated the size difference while human defensin was detrimental to mutant viability. Consistent with the proposed role of MlaA in vesicle biogenesis, the mlaA mutant released 1.7-fold more membrane vesicles. Comparative proteomics of cell envelopes and native membrane vesicles derived from mlaA and wild type bacteria revealed enrichment of TadA-which recodes proteins through mRNA editing-as well as increased levels of adhesins and virulence factors. MlaA-deficient gonococci significantly outcompeted (up to 16-fold) wild-type bacteria in the murine lower genital tract, suggesting the growth advantage or increased expression of virulence factors afforded by inactivation of mlaA is advantageous in vivo. Based on these results, we propose N. gonorrhoeae restricts MlaA levels to modulate cell envelope homeostasis and fine-tune virulence. Author summary The Gram-negative outer membrane is a formidable barrier, primarily because of its asymmetric composition. A layer of lipopolysaccharide is exposed to the external environment and phospholipids are on the internal face of the outer membrane. MlaA is part of a bacterial system that prevents phospholipid accumulation within the lipopolysaccharide layer. If MlaA is removed, membrane asymmetry is disrupted and bacteria become more vulnerable to certain antimicrobials. Neisseria gonorrhoeae causes millions of infections worldwide annually. A growing number are resistant to available antibiotics. Improving our understanding of gonococcal pathogenicity and basic biological processes is required to facilitate the discovery of new weapons against gonorrhea. We investigated the role of MlaA in N. gonorrhoeae and found that when MlaA was absent, bacteria were more sensitive to antibiotics and human defensins. However, the mutant bacteria produced more membrane vesicles-packages of proteins wrapped in membrane material. Mutant vesicles and cell envelopes were enriched in proteins that contribute to disease. These alterations significantly increased mutant fitness during experimental infection of the female mouse genital tract. Our results provide new insights into the processes N. gonorrhoeae uses to fine-tune its ability to stay fit in the hostile environment of the genital tract.