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The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes. Here the authors show that ComFC is a membrane-associated protein that participates in the transport of DNA through the cell membrane and the handling of the single-stranded DNA once delivered into the cytoplasm.

Some bacteria can take up DNA molecules from the environment. Here, Damke et al. identify a DNA-binding protein in Helicobacter pylori that is required for DNA import into the periplasm and that interacts with an inner-membrane channel that translocates the DNA into the cytoplasm.

Release of conserved cytoplasmic proteins is widely spread among Gram-positive and Gram-negative bacteria. Because these proteins display additional functions when located at the bacterial surface, they have been qualified as moonlighting proteins. The GAPDH is a glycolytic enzyme which plays an important role in the virulence processes of pathogenic microorganisms like bacterial invasion and host immune system modulation. However, GAPDH, like other moonlighting proteins, cannot be secreted through active secretion systems since they do not contain an N-terminal predicted signal peptide. In this work, we investigated the mechanism of GAPDH export and surface retention in Streptococcus pneumoniae, a major human pathogen. We addressed the role of the major autolysin LytA in the delivery process of GAPDH to the cell surface. Pneumococcal lysis is abolished in the ΔlytA mutant strain or when 1% choline chloride is added in the culture media. We showed that these conditions induce a marked reduction in the amount of surface-associated GAPDH. These data suggest that the presence of GAPDH at the surface of pneumococcal cells depends on the LytA-mediated lysis of a fraction of the cell population. Moreover, we demonstrated that pneumococcal GAPDH binds to the bacterial cell wall independently of the presence of the teichoic acids component, supporting peptidoglycan as a ligand to surface GAPDH. Finally, we showed that peptidoglycan-associated GAPDH recruits C1q from human serum but does not activate the complement pathway.

Conventional drug design embraces the "one gene, one drug, one disease" philosophy. Polypharmacology, which focuses on multi-target drugs, has emerged as a new paradigm in drug discovery. The rational design of drugs that act via polypharmacological mechanisms can produce compounds that exhibit increased therapeutic potency and against which resistance is less likely to develop. Additionally, identifying multiple protein targets is also critical for side-effect prediction. One third of potential therapeutic compounds fail in clinical trials or are later removed from the market due to unacceptable side effects often caused by off-target binding. In the current work, we introduce a multidimensional strategy for the identification of secondary targets of known small-molecule inhibitors in the absence of global structural and sequence homology with the primary target protein. To demonstrate the utility of the strategy, we identify several targets of 4,5-dihydroxy-3-(1-naphthyldiazenyl)-2,7-naphthalenedisulfonic acid, a known micromolar inhibitor of Trypanosoma brucei RNA editing ligase 1. As it is capable of identifying potential secondary targets, the strategy described here may play a useful role in future efforts to reduce drug side effects and/or to increase polypharmacology.

One of the most abundant DNA lesions induced by Reactive oxygen species (ROS) is 8-oxoG, a highly mutagenic lesion that compromises genetic instability when not efficiently repaired. 8-oxoG is specifically recognized by the DNA-glycosylase OGG1 that excises the base and initiates the Base Excision Repair pathway (BER). Furthermore, OGG1 has not only a major role in DNA repair but it is also involved in transcriptional regulation. Cancer cells are particularly exposed to ROS, thus challenging their capacity to process oxidative DNA damage has been proposed as a promising therapeutic strategy for cancer treatment. Two competitive inhibitors of OGG1 (OGG1i) have been identified, TH5487 and SU0268, which bind to the OGG1 catalytic pocket preventing its fixation to the DNA. Early studies with these inhibitors show an enhanced cellular sensitivity to cytotoxic drugs and a reduction in the inflammatory response. Our study uncovers two unreported off-targets effects of these OGG1i that are independent of OGG1. In vitro and in cellulo approaches have unveiled that OGG1i TH5487 and SU0268, despite an unrelated molecular structure, are able to inhibit some members of the ABC family transporters, in particular ABC B1 (MDR1) and ABC G2 (BCRP). The inhibition of these efflux pumps by OGG1 inhibitors results in a higher intra-cellular accumulation of various fluorescent probes and drugs, and largely contributes to the enhanced cytotoxicity observed when the inhibitors are combined with cytotoxic agents. Furthermore, we found that SU0268 has an OGG1-independent anti-mitotic activity—by interfering with metaphase completion—resulting in a high cellular toxicity. These two off-target activities are observed at concentrations of OGG1i that are normally used for in vivo studies. It is thus critical to consider these previously unreported non-specific effects when interpreting studies using TH5487 and SU0268 in the context of OGG1 inhibition. Additionally, our work highlights the persistent need for new specific inhibitors of the enzymatic activity of OGG1.

ABSTRACT Ovococci form a morphological group that includes several human pathogens (enterococci and streptococci). Their shape results from two modes of cell wall insertion, one allowing division and one allowing elongation. Both cell wall synthesis modes rely on a single cytoskeletal protein, FtsZ. Despite the central role of FtsZ in ovococci, a detailed view of the in vivo nanostructure of ovococcal Z-rings has been lacking thus far, limiting our understanding of their assembly and architecture. We have developed the use of photoactivated localization microscopy (PALM) in the ovococcus human pathogen Streptococcus pneumoniae by engineering spDendra2, a photoconvertible fluorescent protein optimized for this bacterium. Labeling of endogenously expressed FtsZ with spDendra2 revealed the remodeling of the Z-ring's morphology during the division cycle at the nanoscale level. We show that changes in the ring's axial thickness and in the clustering propensity of FtsZ correlate with the advancement of the cell cycle. In addition, we observe double-ring substructures suggestive of short-lived intermediates that may form upon initiation of septal cell wall synthesis. These data are integrated into a model describing the architecture and the remodeling of the Z-ring during the cell cycle of ovococci. IMPORTANCE The Gram-positive human pathogen S. pneumoniae is responsible for 1.6 million deaths per year worldwide and is increasingly resistant to various antibiotics. FtsZ is a cytoskeletal protein polymerizing at midcell into a ring-like structure called the Z-ring. FtsZ is a promising new antimicrobial target, as its inhibition leads to cell death. A precise view of the Z-ring architecture in vivo is essential to understand the mode of action of inhibitory drugs (see T. den Blaauwen, J. M. Andreu, and O. Monasterio, Bioorg Chem 55:27–38, 2014, doi:10.1016/j.bioorg.2014.03.007, for a review on FtsZ inhibitors). This is notably true in ovococcoid bacteria like S. pneumoniae, in which FtsZ is the only known cytoskeletal protein. We have used superresolution microscopy to obtain molecular details of the pneumococcus Z-ring that have so far been inaccessible with conventional microscopy. This study provides a nanoscale description of the Z-ring architecture and remodeling during the division of ovococci.

Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.

Despite years of intensive research, much remains to be discovered to understand the regulatory networks coordinating bacterial cell growth and division. The mechanisms by which Streptococcus pneumoniae achieves its characteristic ellipsoid-cell shape remain largely unknown. In this study, we analyzed the interplay of the cell division paralogs DivIVA and GpsB with the ser/thr kinase StkP. We observed that the deletion of divIVA hindered cell elongation and resulted in cell shortening and rounding. By contrast, the absence of GpsB resulted in hampered cell division and triggered cell elongation. Remarkably, ΔgpsB elongated cells exhibited a helical FtsZ pattern instead of a Z-ring, accompanied by helical patterns for DivIVA and peptidoglycan synthesis. Strikingly, divIVA deletion suppressed the elongated phenotype of ΔgpsB cells. These data suggest that DivIVA promotes cell elongation and that GpsB counteracts it. Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division protein EzrA, which itself interacts with FtsZ. In addition, GpsB interacts directly with DivIVA. These results are consistent with DivIVA and GpsB acting as a molecular switch to orchestrate peripheral and septal PG synthesis and connecting them with the Z-ring via EzrA. The cellular co-localization of the transpeptidases PBP2x and PBP2b as well as the lipid-flippases FtsW and RodA in ΔgpsB cells further suggest the existence of a single large PG assembly complex. Finally, we show that GpsB is required for septal localization and kinase activity of StkP, and therefore for StkP-dependent phosphorylation of DivIVA. Altogether, we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of peptidoglycan (peripheral and septal) synthesis responsible for the pneumococcal ellipsoid cell shape.