Sergio R. Filipe

Spatial and temporal organization of replicating Escherichia coli chromosomes

The positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory‐associated protein have been analysed. Time‐lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage‐specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid‐cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division.

Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus

The cell wall of Staphylococcus aureus is characterized by an extremely high degree of cross-linking within its peptidoglycan (PGN). Penicillin-binding protein 4 (PBP4) is required for the synthesis of this highly cross-linked peptidoglycan. We found that wall teichoic acids, glycopolymers attached to the peptidoglycan and important for virulence in Gram-positive bacteria, act as temporal and spatial regulators of PGN metabolism, controlling the level of cross-linking by regulating PBP4 localization. PBP4 normally localizes at the division septum, but in the absence of wall teichoic acids synthesis, it becomes dispersed throughout the entire cell membrane and is unable to function normally. As a consequence, the peptidoglycan of TagO null mutants, impaired in wall teichoic acid biosynthesis, has a decreased degree of cross-linking, which renders it more susceptible to the action of lysozyme, an enzyme produced by different host organisms as an initial defense against bacterial infection.

Complementation of the Essential Peptidoglycan Transpeptidase Function of Penicillin-Binding Protein 2 (PBP2) by the Drug Resistance Protein PBP2A in Staphylococcus aureus

The essential function of penicillin-binding protein 2 (PBP2) in methicillin-susceptible Staphylococcus aureus RN4220 was clearly established by placing the pbp2 gene under control of the inducible P(spac) promoter; the resulting bacteria were unable to grow in the absence of inducer. In contrast, the deficit in PBP2 caused by inhibition of transcription of the pbp2 gene did not block growth of a methicillin-resistant S. aureus strain expressing the extra penicillin-binding protein PBP2A, a protein of extraspecies origin that is central to the mechanism of methicillin resistance. Several lines of evidence indicate that the essential function of PBP2 that can be compensated for by PBP2A is the transpeptidase activity. This provides direct genetic evidence that PBP2A has transpeptidase activity.

Staphylococcus aureus PBP4 Is Essential for β-Lactam Resistance in Community-Acquired Methicillin-Resistant Strains

ABSTRACT Recent cases of infections caused by community-acquired methicillin-resistant Staphylococcus aureus (MRSA) (CA-MRSA) strains in healthy individuals have raised concerns worldwide. CA-MRSA strains differ from hospital-acquired MRSAs by virtue of their genomic background and increased virulence in animal models. Here, we show that in two common CA-MRSA isolates, USA300 and MW2 (USA400), a loss of penicillin binding protein 4 (PBP4) is sufficient to cause a 16-fold reduction in oxacillin and nafcillin resistance, thus demonstrating that mecA, encoding PBP2A, is not the sole determinant of methicillin resistance in CA-MRSA. The loss of PBP4 was also found to severely affect the transcription of PBP2 in cells after challenge with oxacillin, thus leading to a significant decrease in peptidoglycan cross-linking. Autolysis, which is commonly associated with the killing mechanism of penicillin and β-lactams, does not play a role in the reduced resistance phenotype associated with the loss of PBP4. We also showed that cefoxitin, a semisynthetic β-lactam that binds irreversibly to PBP4, is synergistic with oxacillin in killing CA-MRSA strains, including clinical CA-MRSA isolates. Thus, PBP4 represents a major target for drug rediscovery against CA-MRSA, and a combination of cefoxitin and synthetic penicillins may be an effective therapy for CA-MRSA infections.

Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo

We report an efficient, controllable, site‐specific replication roadblock that blocks cell proliferation, but which can be rapidly and efficiently reversed, leading to recovery of viability. Escherichia coli replication forks of both polarities stalled in vivo within the first 500 bp of a 10 kb repressor‐bound array of operator DNA‐binding sites. Controlled release of repressor binding led to rapid restart of the blocked replication fork without the participation of homologous recombination. Cytological tracking of fork stalling and restart showed that the replisome‐associated SSB protein remains associated with the blocked fork for extended periods and that duplication of the fluorescent foci associated with the blocked operator array occurs immediately after restart, thereby demonstrating a lack of sister cohesion in the region of the array. Roadblocks positioned near oriC or the dif site did not prevent replication and segregation of the rest of the chromosome.

Requirements of peptidoglycan structure that allow detection by the Drosophila Toll pathway

The Drosophila immune system is able to discriminate between classes of bacteria. Detection of Gram‐positive bacteria involves a complex of two pattern recognition receptors: peptidoglycan recognition protein SA (PGRP‐SA) and Gram‐negative binding protein 1 (GNBP1). These activate the Toll signalling pathway. To define the cell wall components sensed by the host, we used highly purified peptidoglycan fragments of two principal Gram‐positive bacterial pathogens Staphylococcus aureus and Streptococcus pneumoniae. We report that in both peptidoglycans, the minimal structure needed to activate the Toll pathway is a muropeptide dimer and that the free reducing end of the N‐acetyl muramic acid residues of the muropeptides is essential for activity. Monomeric muropeptides were inactive and inhibitory in combination with dimers. Finally, peptidoglycan was degraded by the haemolymph of wild‐type but not GNBP1 mutant flies. We suggest a model whereby GNBP1 is involved in the hydrolysis of Gram‐positive peptidoglycan producing new glycan reducing ends, which are subsequently detected by PGRP‐SA.

Sensing of Gram‐positive bacteria in Drosophila: GNBP1 is needed to process and present peptidoglycan to PGRP‐SA

Genetic evidence indicates that Drosophila defense against Gram‐positive bacteria is mediated by two putative pattern recognition receptors acting upstream of Toll, namely Gram‐negative binding protein 1 (GNBP1) and peptidoglycan recognition protein SA (PGRP‐SA). Until now however, the molecular recognition proceedings for sensing of Gram‐positive pathogens were not known. In the present, we report the physical interaction between GNBP1 and PGRP‐SA using recombinant proteins. GNBP1 was able to hydrolyze Gram‐positive peptidoglycan (PG), while PGRP‐SA bound highly purified PG fragments (muropeptides). Interaction between these proteins was enhanced in the presence of PG or muropeptides. PGRP‐SA binding depended on the polymerization status of the muropeptides, pointing to constraints in the number of PGRP‐SA molecules bound for signaling initiation. We propose a model whereby GNBP1 presents a processed form of PG for sensing by PGRP‐SA and that a tripartite interaction between these proteins and PG is essential for downstream signaling.

Fluorescence Ratio Imaging Microscopy Shows Decreased Access of Vancomycin to Cell Wall Synthetic Sites in Vancomycin-Resistant Staphylococcus aureus

ABSTRACT A new method of fluorescence ratio imaging microscopy was used to compare the in vivo binding capacity and the access of a fluorescent derivative of vancomycin to the cell wall synthetic sites in isogenic pairs of vancomycin-susceptible and -resistant laboratory mutants and vancomycin-intermediate and -susceptible clinical isolates of Staphylococcus aureus. Live cells of resistant strains were found to bind approximately 1.5 times more antibiotic, but there was no correlation between the increased binding capacity and the MICs of the strains. In both susceptible and resistant bacteria, the subcellular sites of wall synthesis were localized to the division septa, but the rate of diffusion of drug molecules to these sites was reduced in resistant cells. The findings allow a reinterpretation of the mechanism of vancomycin resistance in which the path of vancomycin to its lethal target (lipid II) is considered to be through the division septum and therefore is dependent on the stage of the staphylococcal cell cycle.

Cell shape dynamics during the staphylococcal cell cycle

Staphylococcus aureus is an aggressive pathogen and a model organism to study cell division in sequential orthogonal planes in spherical bacteria. However, the small size of staphylococcal cells has impaired analysis of changes in morphology during the cell cycle. Here we use super-resolution microscopy and determine that S. aureus cells are not spherical throughout the cell cycle, but elongate during specific time windows, through peptidoglycan synthesis and remodelling. Both peptidoglycan hydrolysis and turgor pressure are required during division for reshaping the flat division septum into a curved surface. In this process, the septum generates less than one hemisphere of each daughter cell, a trait we show is common to other cocci. Therefore, cell surface scars of previous divisions do not divide the cells in quadrants, generating asymmetry in the daughter cells. Our results introduce a need to reassess the models for division plane selection in cocci.

The murMN operon: A functional link between antibiotic resistance and antibiotic tolerance in Streptococcus pneumoniae

Inactivation of the recently identified murMN operon in penicillin-resistant strains of Streptococcus pneumoniae was shown already to cause two major effects: elimination of branched-structured muropeptides from the cell wall and complete loss of penicillin resistance. We now show that cells with inactivated murMN also have a third phenotype: an increased susceptibility to lysis when exposed to low concentrations of fosfomycin, d-cycloserine, vancomycin, and nisin, indicating a wide-spectrum hypersensitivity to inhibitors of both early and late stages of cell wall biosynthesis. Mutants of murMN also lysed faster than the parental strain when treated with the detergent deoxycholate. Several different alleles of murM cloned in plasmid pLS578 and introduced into a murM deletion mutant of the penicillin-resistant strain Pen6 were able to reconstitute each one of the three mutant phenotypes: the highly branched cell wall structure, original high level of penicillin resistance, and normal sensitivity to lysis. In a penicillin-susceptible strain the same experiments caused increased concentration of cell wall branched peptides and suppression of sensitivity to antibiotic induced lysis. The observations suggest that the murMN operon plays a key role in the regulation of a stress-response pathway that can be triggered by perturbation of cell wall biosynthesis in S. pneumoniae.