Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans

The Toll and Imd pathways are not required for Wolbachia-mediated dengue virus interference

Wolbachia blocks dengue virus replication in Drosophila melanogaster as well as in Aedes aegypti. Using the Drosophila model and mutations in the Toll and Imd pathways, we showed that neither pathway is required for expression of the dengue virusblocking phenotype in the Drosophila host. This provides additional evidence that the mechanistic basis of Wolbachia-mediated dengue virus blocking in insects is more complex than simple priming of the host insect innate immune system

Characterization of a Broadly Neutralizing Monoclonal Antibody against SARS-CoV-2 Variants

10.3390/v14020230VIRUSES-BASEL14

Enhanced stability of the SARS CoV-2 spike glycoprotein following modification of an alanine cavity in the protein core.

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3-4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala1016 and Ala1020 in the 3-4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala1016/Ala1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID50s) in the range 2,700-5,110 for ancestral and Delta-derived viruses, and 210-1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines

Blood-feeding and <i>Wolbachia</i> densities in mosquito heads and bodies.

<p>Bodies (A) and heads (B) of outbred laboratory <i>w</i>Mel (MGYP2.out) and field-released <i>w</i>Mel (<i>w</i>Mel.F) <i>A. aegypti</i> at days 7 and 14 post blood-feeding. Bars denote medians. P&lt;0.05 (*), P&lt;0.01 (**), P&lt;0.001 (***). Each point represents an individual mosquito.</p

Rates of infection (%) for three DENV strains among field (<i>w</i>Mel.F) and laboratory <i>Wolbachia</i>-infected (MGYP2.out) and uninfected (wildtype) mosquito lines at days 7 and 14 post-infection (p.i.) (experiment 2).

<p>Adjusted Fisher's exact test p-values&lt;0.05 (*), &lt;0.001 (***). P-values shown refer to comparisons between wildtype and <i>w</i>Mel.F mosquitoes.</p

Localization of <i>Wolbachia</i> in different <i>A. aegypti</i> tissues visualized using FISH.

<p>Outbred laboratory <i>w</i>Mel (MGYP2.out) (A, C, G, E) and field-released <i>w</i>Mel (<i>w</i>Mel.F) (B, D, F, H) mosquitoes at day 7 post DENV infection. <i>Wolbachia</i> stained in red (Alexa 594) and cell nuclei in blue (DAPI). Images are representative of 4–5 mosquitoes per line. Bars represent 50 µM scale.</p

Blood-feeding and <i>Wolbachia</i> densities in whole mosquitoes.

<p>Outbred laboratory <i>w</i>Mel (MGYP2.out) and field-released <i>w</i>Mel (<i>w</i>Mel.F) <i>A. aegypti</i> at 7 (A) and 14 (B) days post blood-feeding (BF) versus non-blood fed (NBF) controls. Bars denote medians. P&lt;0.05 (*), P&lt;0.01 (**), P&lt;0.001 (***). Each point represents an individual mosquito.</p

Experiment 2: DENV replication in wildtype, outbred laboratory <i>w</i>Mel (MGYP2.out) and field-released <i>w</i>Mel (<i>w</i>Mel.F) <i>A. aegypti</i>.

<p>DENV replication in bodies (A) and heads (B) of mosquitoes challenged with three strains (DENV2-92T, DENV1-P307, DENV3-Cairns08/09), assayed at 14 days post-infection. DENV levels determined using one-step qRT-PCR and expressed as copies per 1 µg of total RNA. Bars denote medians. P&lt;0.05 (*), P&lt;0.01 (**), P&lt;0.001 (***). Each point represents an individual mosquito.</p

Experiment 1: DENV replication in wildtype and field-released (<i>w</i>Mel.F) <i>A. aegypti</i>.

<p>DENV replication in bodies (A) and heads (B) of mosquitoes challenged with three strains (DENV2-92T, DENV1-P249, DENV2-P410), assayed at 14 days post-infection. DENV levels determined using one-step qRT-PCR and expressed as copies per 1 µg of total RNA. Bars denote medians. P&lt;0.05 (*), P&lt;0.01 (**), P&lt;0.001 (***). Each point represents an individual mosquito.</p