Neisseria gonorrhoeae MlaA influences gonococcal virulence and membrane vesicle production

Abstract

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 ΔmlaA mutant released 1.7-fold more membrane vesicles. Comparative proteomics of cell envelopes and native membrane vesicles derived from ΔmlaA 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.

Structural insight into outer membrane asymmetry maintenance of Gram-negative bacteria by the phospholipid transporter MlaFEDB

“…On the other hand, the molecular mechanism of how PLs are trafficked between the IM and the OM to maintain the OM asymmetry remains unclear. The maintenance of lipid asymmetry (Mla) pathway is believed to be important for maintaining the asymmetry and integrity of the OM by trafficking PLs between the IM and the OM [35][36][37][38][39][40][41] . Mutants of the Mla pathway in Escherichia coli (E. coli) or other pathogenic species show OM permeability and integrity defects and increased susceptibility to antibiotics [36][37][38][39][40][41][42] .…”

“…The maintenance of lipid asymmetry (Mla) pathway is believed to be important for maintaining the asymmetry and integrity of the OM by trafficking PLs between the IM and the OM [35][36][37][38][39][40][41] . Mutants of the Mla pathway in Escherichia coli (E. coli) or other pathogenic species show OM permeability and integrity defects and increased susceptibility to antibiotics [36][37][38][39][40][41][42] . Six proteins are involved in the Mla pathway, including an ATP-binding cassette (ABC) transporter MlaFE associated with an auxiliary periplasmic membrane protein MlaD and an auxiliary cytoplasmic protein MlaB, a periplasmic protein MlaC and an OM protein MlaA associated with an osmoporin OmpF/C 36,43,44 (Figure 1A).…”

Comparative Proteomics of Extended-Spectrum Cephalosporin-Resistant Neisseria gonorrhoeae Isolates Demonstrates Altered Protein Synthesis, Metabolism, Substance Transport, and Membrane Permeability

“…Normalized abundance was calculated from the raw abundance divided by the normalization factor. Differentially expressed proteins were filtered if their fold change (FC) was over 1.5 or below −1.5 as compared to ATCC49226 and contained at least 2 unique peptides (P < 0.01) (Baarda et al, 2019;El-Rami et al, 2019).…”

Evolution toward maximum transport capacity of the Ttg2 ABC system inPseudomonas aeruginosa

“…MlaC would then transport the phospholipids across the periplasm and deliver them to MlaD for active internalization through the IM 23 . Deletion of the genes of this system is known to destabilize the OM, and bacterial strains lacking any of the Mla components are more susceptible to membrane stress agents 19, 24, 25, 26, 27, 28, 29, 30 . More recently, the retrograde transport hypothesis has been questioned and a new role for this system in anterograde phospholipid transport has been suggested 29, 31 .…”