msi in the news

march 2017 edition

In vitro reconstitution demonstrates the cell wall ligase activity of LCP proteins

The bacterial cell wall in gam positive bacteria is made up of peptidoglycan (PG) as well as glycopolymers – specifically wall teichoic acid (WTA) in Staphylococcus aureus – but previously the enzyme responsible for linking the two was not verified. Genes in the LCP family had been previously implicated in Bacillus subtilis, but their ligase activity was not proven. A recent paper by Kaitlin Schaefer of the MSI affiliated Kahne and Walker labs showed that enzymes in the LCP family show ligase activity in vitro in S. aureus. Short WTA precursors and peptidoglycan oligomers were synthesized chemically, and purified LcpA, LcpB, and LcpC transferred the radiolabeled WTA precursors to the PG oligomers. Ligation was confirmed using high resolution mass spectrometry. These three enzymes are not redundant, however. Plating single and double mutants informed by a screen of a transposon mutant library, the study found that strains without lcpB and lcpC were viable plated on amasacrine, which inhibits DltB, necessary for d-alaynylation. Together, lack of D-alanylation and an absence of WTA are synthetically lethal, and when lcpA is deleted, strains are not viable, implying that this enzyme is most necessary for WTA ligation. Interestingly, MRSA strains, which encode mecA, a gene for beta lactam-resistant penicillin binding protein, also require tarO, the first gene in the WTA pathway. When lcpA is deleted in these strains, they become resensitized to beta lactam oxacillin.

Figure 1: a) Scheme for enzymatic synthesis of WTA precursors LIIAWTA and LIIBWTA from synthetic LIWTA. Radiolabels were optionally incorporated using UDP-[14C]GlcNAc for LIIAWTA and CDP-[14C]glycerol for LIIBWTA. b) Scheme for enzymatic synthesis of PG oligomers from synthetic Lipid II, which optionally contained [14C]GlcNAc. SgtB* contains a Y181D substitution and produces short PG oligomers (n = 1–7 disaccharide repeats). c) An autoradiograph showing that LcpA transfers [14C]WTA precursors to cold PG oligomers (lanes 2 and 4). Control lanes 5 and 6 contain [14C]Lipid II and [14C]PG oligomers, respectively. Data represents experiments performed with a minimum of three replicates.

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Global and local selection acting on the pathogen Stenotrophomonas maltophilia in the human lung

A recent paper by Hattie Chung, a graduate student in the MSI affiliated Kishony lab, investigated the populations in the lung of a cystic fibrosis patient who underwent bilateral lung transplantation. Sputum and tissue specimens from the lung were collected, and 552 genomes of Strenotrophomonas maltophilia, an emerging CF pathogen, were sequenced. The study shows that these genomes were comprised of major and minor lineages, A and S, and B and C respectively. These lineages were separated by 47 SNPs and copy number variations in four gene regions, overall an indication of diversification of the pathogen. Furthermore, the results indicated positive selection, as the nonsynonymous to synonymous mutation ratio was high, indicated by a dN/dS of 1.9. Genes for virulence contained many lineage-separating mutations. Four genes had at least two lineage-separating mutations, and two of these are implicated in antibiotic resistance. These findings indicate the presence of a selective pressure, for example the three different drug classes administered to the infected patient. From a spatial perspective, the lineages coexisted in most sites. Major lineages but not minor lineages were shown in the sputum sample. Some sites on the lung were dominated by only one of the major lineages though; despite general dispersion, some strains were enriched in certain sites. Specific sites also exert unique selective pressures. For example, mutations in a merC homologue, which occurred independently in both lineages, proliferated at the same locations.

Figure 2: a) Parsimony tree of the population constructed from 334 polymorphic mutations. Most recent common ancestor (MRCA) on the left. The mutations of eight genes with recurrent mutations are indicated on the tree, each gene with its own symbol. Lineages A and S (small colony variant) form the majority of the population. b) Resistance profile of all isolates against three antibiotics used in treatment, in twofold drug concentrations. Each row is an isolate aligned to its position on the phylogeny. NA, not available; 0, isolate grew only on no drug plates. The differences in resistance between lineages A and S were highly significant: ceftazidime P=4 × 10^-39, ciprofloxacin P=3 × 10^-43, tobramycin P=2 × 10^-34, Kolmogorov–Smirnov test. c) Lineage-separating mutations are under positive selection (green; dN/dS=1.9, P=0.027, 95% confidence interval (CI) 1.06–3.70), whereas within-lineage mutations are neutral (white; dN/dS=0.93).

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Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform

CRISPR interference (CRISPRi) is an incredible tool for identifying drug targets in pathogenic bacteria. However, CRISPR had been unsuccessful for use in mycobacteria due to its poor efficiency and proteotoxicity; specifically the dCas9 from Streptococcus pyogenes, or dCas9Spy, gets partially degraded in mycobacteria and also sensitizes them to stress. A recent study by Jeremy Rock in the MSI-affiliated Fortune lab optimized the CRISPRi system using a dCas9 from Streptococcus thermophilus, or dCas9Sth1, and showed it to be robust in both Mycobacterium smegmatis and Mycobacterium tuberculosis. This particular Cas9 variant was found in a screen of 11 cas9 orthologues identified fro their small size and specific crRNA, tracrRNA, and PAM sequences. Of these, dCas9Sth1 was the most robust gene silencer, as expressed in M. smegmatis and M. tuberculosis on a plasmid under an anhydrotetracycline (ATC) inducible promoter. Furthermore, this enzyme can target with non-canonical PAM sequences, or the sequences just downstream of the targeted DNA sequence that are necessary for helping Cas9 effectively localize. Interestingly, the system can be used far from transcriptional start sites. This would found when researchers used sgRNAs along the genes in the pptT and groES-groEL operons. They found no correlation in repression efficacy with the targeting site distance from the transcriptional start site, but did notice a downstream polar effect: any genes downstream of the silenced gene would also be silenced. In M. smegmatis, the optimized CRISPRi system was used to show that partial knockdown of folA, folP1, and folC sensitized the bacteria to antibiotics.

Figure 3: a) dCas9Spy can mediate high-level knockdown of endogenous targets in M. smegmatis. sgRNAs targeting dnaE1 were co-expressed with dCas9Spy or dCas9Sth1 (+ATc). Gene knockdown was quantified by qRT–PCR; the consequences of dnaE1 knockdown were monitored by spotting dilutions of each culture on the indicated media. b) sgRNAs targeting pptT in M. smegmatis were co-expressed with the indicated dCas9 protein (+ATc) and plated as in a. c) The indicated dCas9 proteins were haemagglutinin (HA)-tagged, co-expressed with a non-targeting control sgRNA (+ATc) and monitored by western blot. RpoB protein levels are shown as loading controls. *Background anti-HA cross-reactive band. d) dCas9Spy sensitizes M. smegmatis to sub-minimum inhibitory concentration (sub-MIC) drug treatment. Non-targeting control sgRNAs were co-expressed with dCas9Spy or dCas9Sth1 (+ATc); the consequences of dCas9 expression in the presence of sub-MIC concentrations of the indicated drugs were monitored by spotting serial dilutions of each culture as in a. Approximately 5,000 cells are deposited in the first spot and each subsequent spot is a tenfold serial dilution. AMK, amikacin; NAT, nourseothricin; STR, streptomycin; RIF, rifampicin.

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