Molecular Markers of In Vivo Plasmodium vivax Resistance to Amodiaquine Plus Sulfadoxine-Pyrimethamine: Mutations in pvdhfr and pvmdr1

Background. Molecular markers for sulfadoxine-pyrimethamine (SP) resistance in Plasmodium vivax have been reported. However, data on the molecular correlates involved in the development of resistance to 4-aminoquinolines and their association with the in vivo treatment response are scarce. Methods. We assessed pvdhfr (F57L/I, S58R, T61M, S117T/N, and I173F/L) and pvmdr1 (Y976F and F1076L) mutations in 94 patients who received amodiaquine (AQ) plus SP in Papua New Guinea (PNG). We then investigated the association between parasite genotype and treatment response. Results. The treatment failure (TF) rate reached 13%. Polymorphisms in pvdhfr F57L, S58R, T61M, and S117T/N and in pvmdr1 Y976F were detected in 60%, 67%, 20%, 40%, and 39% of the samples, respectively. The single mutant pvdhfr 57 showed the strongest association with TF (odds ratio [OR], 9.04; P=.01). The combined presence of the quadruple mutant pvdhfr 57L+58R+61M+117T and pvmdr1 mutation 976F was the best predictor of TF (OR, 8.56; P=.01). The difference in TF rates between sites was reflected in the genetic drug-resistance profile of the respective parasites. Conclusions. The present study identified a new molecular marker in pvmdr1 that is associated with the in vivo response to AQ+SP. We suggest suitable marker sets with which to monitor P. vivax resistance against AQ+SP in countries where these drugs are use

SecA localization and SecA-dependent secretion occurs at new division septa in group B Streptococcus.

International audienceExported proteins of Streptococcus agalactiae (GBS), which include proteins localized to the bacterial surface or secreted into the extracellular environment, are key players for commensal and pathogenic interactions in the mammalian host. These proteins are transported across the cytoplasmic membrane via the general SecA secretory pathway and those containing the so-called LPXTG sorting motif are covalently attached to the peptidoglycan by sortase A. How SecA, sortase A, and LPXTG proteins are spatially distributed in GBS is not known. In the close relative Streptococcus pyogenes, it was shown that presence of the YSIRKG/S motif (literally YSIRKX3Gx2S) in the signal peptide (SP) constitutes the targeting information for secretion at the septum. Here, using conventional and deconvolution immunofluorescence analyses, we have studied in GBS strain NEM316 the localization of SecA, SrtA, and the secreted protein Bsp whose signal peptide contains a canonical YSIRKG/S motif (YSLRKykfGlaS). Replacing the SP of Bsp with four other SPs containing or not the YSIRKG/S motif did not alter the localized secretion of Bsp at the equatorial ring. Our results indicate that secretion and cell wall-anchoring machineries are localized at the division septum. Cell wall- anchored proteins displayed polar (PilB, Gbs0791), punctuate (CspA) or uniform distribution (Alp2) on the bacterial surface. De novo secretion of Gbs0791 following trypsin treatment indicates that it is secreted at the septum, then redistributed along the lateral sides, and finally accumulated to the poles. We conclude that the ±YSIRK SP rule driving compartimentalized secretion is not true in S. agalactiae

Molecular Dissection of the secA2 Locus of Group B Streptococcus Reveals that Glycosylation of the Srr1 LPXTG Protein Is Required for Full Virulence▿ †

In streptococci, the secA2 locus includes genes encoding the following: (i) the accessory Sec components (SecA2, SecY2, and at least three accessory secretion proteins), (ii) two essential glycosyltranferases (GTs) (GtfA and GtfB), (iii) a variable number of dispensable additional GTs, and (iv) a secreted serine-rich LPXTG protein which is glycosylated in the cytoplasm and transported to the cell surface by this accessory Sec system. The secA2 locus of Streptococcus agalactiae strain NEM316 is structurally related to those found in other streptococci and encodes the serine-rich surface protein Srr1. We demonstrated that expression of Srr1 but not that of the SecA2 components and the associated GTs is regulated by the standalone transcriptional regulator Rga. Srr1 is synthesized as a glycosylated precursor, secreted by the SecA2 system, and anchored to the cell wall by the housekeeping sortase A. Srr1 was localized preferentially at the old poles. GtfA and/or GtfB, but not the six additional GTs, is essential for the production of Srr1. These GTs are involved in the attachment of GlcNac and sialic acid to Srr1. Full glycosylation of Srr1 is associated with the cell surface display of a protein that is more resistant to proteolytic attack. Srr1 contributes to bacterial adherence to human epithelial cell lines and virulence in a neonatal rat model. The extent of Srr1 glycosylation by GtfC to -H modulates bacterial adherence and virulence

Surface distribution of Bsp in <i>S.</i><i>agalactiae</i> NEM316 and derivatives.

<p>Bacteria were harvested in exponential phase (OD₆₀₀ = 0.3) and labeled with DAPI (blue) plus rabbit anti-Bsp pAb (green). The bacterial strains studied were: NEM316 (WT) (positive control); NEM316Δ<i>bsp</i>/pTCV-erm without insert (negative control); NEM316Δ<i>bsp</i>/pTCV-<i>erm</i> expressing recombinant Bsp with the signal peptide of CspA, Alp2, PilB, or Gbs0791. Data are representative of three independent experiments.</p

Conventional immunofluorescence microscopy showing localization of SecA in the non-capsulated mutant Δ<i>cpsE</i> compared to the parental WT NEM316.

<p>(A) Immunofluorescence microscopy of bacteria harvested in mid-exponential phase and visualized with fluorescent vancomycin (green) or plus rabbit anti-SecA pAb (red). Note that SecA is more concentrated in the constricting septa and its neighboring region in a pattern very similar to that reported for the non-capsulated strain of <i>S. pneumoniae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065832#pone.0065832-Tsui1" target="_blank">[31]</a>. (B) Immunofluorescence of bacteria harvested in mid-exponential phase and visualized with rabbit pAb against Bsp and PilB.</p

Subcellular localization of Bsp and CAMP factor in <i>S.</i><i>agalactiae</i> NEM316.

<p>(A, B) IFM images of bacteria harvested in exponential phase (OD₆₀₀ = 0.3) and labeled with specific antibodies directed against Bsp (A) or CAMP factor (B) revealed with AlexaFluor 594- fluorescent secondary antibody (red). Outline of the cells was visualized by DIC and active zone of peptidoglycan synthesis with fluorescent vancomycin (green). (C) Signal peptides of Bsp and CAMP proteins. The amino acids constituting the YSIRK motif are highlighted in red. Schematic localization of vancomycin, Bsp, and CAMP factor at non-constricting septa.</p

Expression in GBS of Bsp recombinant proteins with structurally unrelated signal peptides.

<p>(A) <i>Bam</i>HI-<i>Not</i>I PCR fragments carrying the ribosome binding site (RBS) and the signal peptides (SP) of 5 SecA-dependent substrates (Bsp, Alp2, Gbs0791, PilB, and CspA) were fused in frame with a <i>Not</i>I-<i>Pst</i>I PCR fragment coding the Bsp protein devoid of its signal peptide (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065832#pone.0065832.s004" target="_blank">Table S1</a>). The resulting <i>Bam</i>HI-<i>Pst</i>I fragments were cloned downstream the constitutive P<i>tetM</i> promoter from the low-copy-number pTCV<i>-erm</i>. The SP and Bsp sequences are indicated in upper-bold italic characters and upper-bold characters, respectively. The boxed RP motif in all proteins corresponded to the translation of the two internal codons of the <i>Not</i>I restriction site (CGGCCG). All but one SP were predicted with SignalP 4.1 (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">www.cbs.dtu.dk/services/SignalP/</a>) whereas the remaining (Alp2) was predicted with PrediSi (<a href="http://www.predisi.de" target="_blank">www.predisi.de</a>). The AA residues in the SP thought to direct localized secretion at the bacterial surface are indicated in red characters. Arrowheads indicate the predicted site of cleavage of the various SP. (B) Analysis of surface display of Bsp recombinant proteins in a Δ<i>bsp</i> mutant strain by immunoblotting. Whole bacterial cells harvested in exponential (OD₆₀₀ 0.3) or stationary (OD₆₀₀ 1.2) phases were washed, resuspended in phosphate buffer saline to similar density and spotted on nitrocellulose. Membranes were hybridized with specific anti-Bsp antibodies or with anti-GBS pAb (loading control). (C) Western blotting analysis of culture supernatants. Proteins were separated on 4–12% gradient Tris-acetate Criterion XT SDS-PAGE gel, then transferred onto a nitrocellulose membrane, and detected by immunoblotting with specific anti-Bsp and anti-CAMP antibodies. In (B) and (C), the Δ<i>bsp</i> mutant strain harboring pTCV-<i>erm</i> (negative control) or pTCV-<i>erm</i> directing synthesis of recombinant Bsp proteins associated with Alp2, Gbs0791, PilB, and CspA signal peptides were used.</p

Surface localization of SrtA and unrelated cell wall-anchored proteins in <i>S.</i><i>agalactiae</i> NEM316.

<p>(A, B) Bacteria harvested in exponential phase (OD₆₀₀ = 0.3) were labeled with (A) DAPI (blue) or guinea pig anti-SrtA pAb (green) and (B) with rabbit pAb against PilB, Gbs0791, Alp2, or CspA (red).</p

Distribution of SecA at the surface of <i>S.</i><i>agalactiae</i> (GBS NEM316) and <i>S. pyogenes</i> (GAS M18).

<p>(A) Western blot image showing that the polyclonal antibody directed against <i>E. coli</i> SecA recognizes a band of approximately 90 kDa in both GAS M18 and GBS NEM316 strains. (B) Conventional immunofluorescence microscopy showing the differential distribution of SecA at the surface of GBS NEM316 versus GAS strain M18 collected in exponential and stationary phase of growth. Heterogeneity of SecA distribution was quantified by eye following analysis of randomly selected fields.</p