Abstract

The β-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Given its role in the production of OMPs for survival and pathogenesis, BAM represents an attractive target for the development of therapeutic interventions, including drugs and vaccines against multidrug-resistant bacteria such as Neisseria gonorrhoeae The first structure of BamA, the central component of BAM, was from N. gonorrhoeae, the etiological agent of the sexually transmitted disease gonorrhea. To aid in pharmaceutical targeting of BAM, we expanded our studies to BamD and BamE within BAM of this clinically relevant human pathogen. We found that the presence of BamD, but not BamE, is essential for gonococcal viability. However, BamE, but not BamD, was cell-surface-displayed under native conditions; however, in the absence of BamE, BamD indeed becomes surface-exposed. Loss of BamE altered cell envelope composition, leading to slower growth and an increase in both antibiotic susceptibility and formation of membrane vesicles containing greater amounts of vaccine antigens. Both BamD and BamE are expressed in diverse gonococcal isolates, under host-relevant conditions, and throughout different phases of growth. The solved structures of Neisseria BamD and BamE share overall folds with Escherichia coli proteins but contain differences that may be important for function. Together, these studies highlight that, although BAM is conserved across Gram-negative bacteria, structural and functional differences do exist across species, which may be leveraged in the development of species-specific therapeutics in the effort to combat multidrug resistance.

Highlights

  • The ␤-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of ␤-barrel outer membrane proteins (OMPs) in Gram-negative bacteria

  • Conservation of BamE and BamD was analyzed by comparing DNA sequences between 42,412 Neisseria isolates deposited to the PubMLST database as of April 18, 2017 and demonstrated the presence of 179 alleles with 89 single nucleotide polymorphisms (SNPs) for BamE and 269 alleles with 186 SNPs for BamD (Fig. S2)

  • Using a quantitative proteomic approach, we have previously identified BamAGC, BamDGC, and BamEGC in both cell envelopes and naturally released membrane vesicles derived from four different N. gonorrhoeae strains [7, 55]

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Summary

Results

BLAST searches for the gonococcal homolog of BamE protein, BamEGC, using the genome sequence of N. gonorrhoeae strain FA1090 and sequence comparisons with Clustal Omega (1.2.; http://www.clustal.org/omega/5; Ref. 76) resulted in identification of locus NGO1780 with 28.7 and 92.7% sequence identity to the E. coli and N. meningitidis MC58 counterparts, respectively (Fig. S1). Analysis of SNPs showed high conservation of both BamEGC and BamDGC among different N. gonorrhoeae isolates (Fig. S2) Corroborating this observation, the anti-BamEGC and antiBamDGC antiserum cross-reacted with whole-cell lysates derived from common laboratory strains (FA1090, F62, MS11, FA19, and 1291) and temporally and geographically diversified clinical isolates, including the 2016 World Health Organization (WHO) reference strains (Fig. 3C). These experiments demonstrated that, to BamAGC [8], the accessory lipoproteins BamEGC and BamDGC are ubiquitously expressed in a highly diverse pool of gonococcal isolates, fur-. Subproteome fractions isolated from wildtype N. gonorrhoeae FA1090 were separated by SDS-PAGE and probed with antisera against BAM

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Discussion
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