Pereira, A. R. | bioRxiv Science

bioRxiv2024

Arp2/3 iso-complexes limit t-tubule overgrowth to ensure myofiber formation and synchronous contraction

Muscle cells form a network of plasma membrane invaginations called t-tubules that control calcium release from the endoplasmic reticulum at the triads during muscle contraction. Although the importance of t-tubules for muscle physiology is well established, and abnormalities are found in multiple disorders, the mechanisms that mediate t-tubule growth are unknown. We show that the actomyosin cortex beneath the plasma membrane, regulated by Arp2/3 complexes containing Arpc5, acts as a gatekeeper for the membrane availability required for t-tubule growth. Enlarged t-tubules are formed upon disruption of Arpc5, impairing the synchronisation between plasma membrane depolarization and calcium release. Additionally, ablation of Arpc5 postnatally in myofibers results in muscle fatigue and t-tubule abnormalities, as observed in muscle disorders. We propose that the actomyosin cortex impacts muscle function, offering a potential pathophysiological mechanism for muscle disorders.

cell biology↗

bioRxiv2019

SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in Staphylococcus aureus

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely through the localisation of the late stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and the bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in cell elongation and cell division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 likely constitute cognate pairs of interacting proteins. Lack of RodA-PBP3 decreased cell eccentricity due to deficient pre-septal PGN synthesis, whereas the depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilising the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings/arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate lateral and septal PGN incorporation, respectively, and that the activity of these pairs must be balanced in order to maintain coccoid morphology.

microbiology↗

bioRxiv2018

The ClpX chaperone controls the Staphylococcus aureus cell cycle but can be bypassed by β-lactam antibiotics

The worldwide spread of Staphylococcus aureus strains resistant to almost all {beta}-lactam antibiotics is of major clinical concern. {beta}-lactams interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Here we show that sub-lethal doses of {beta}-lactams stimulate the growth of S. aureus mutants lacking the widely conserved chaperone ClpX. S. aureus clpX mutants have a severe growth defect at temperatures below 37{degrees}C, and we reasoned that a better understanding of this growth defect could provide novel insights into how {beta}-lactam antibiotics interfere with growth of S. aureus. We demonstrate that ClpX is important for coordinating the S. aureus cell cycle, and that S. aureus cells devoid of ClpX fail to divide, or lyze spontaneously, at high frequency unless {beta}-lactams are added to the growth medium. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30{degrees}C, but not at 37{degrees}C. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, {beta}-lactams restored septum synthesis and prevented premature autolytic splitting of clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with {beta}-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, underscoring a functional link between the PBP activity and WTA biosynthesis. The finding that {beta} -lactams can prevent lysis and restore septum synthesis of a mutant with dysregulated cell division lends support to the idea that PBPs function as coordinators of cell division and that {beta} -lactams do not kill S. aureus simply by weakening the cell wall.\n\nAuthor SummaryThe bacterium Staphylococcus aureus is a major cause of human disease, and the rapid spread of S. aureus strains that are resistant to almost all {beta}-lactam antibiotics has made treatment increasingly difficult. {beta}-lactams interfere with cross-linking of the bacterial cell wall but the killing mechanism of this important class of antibiotics is still not fully understood. Here we provide novel insight into this topic by examining a defined S. aureus mutant that has the unusual property of growing markedly better in the presence of {beta}-lactams. Without {beta}-lactams this mutant dies spontaneously at a high frequency due to premature separation of daughter cells during cell division. Cell death of the mutant can, however, be prevented either by exposure to {beta}-lactam antibiotics or by inhibiting synthesis of wall teichoic acid, a major component of the cell wall in Gram-positive bacteria with a conserved role in activation of autolytic splitting of daughter cells. The finding that the detrimental effect of {beta}-lactam antibiotics can be reversed by a mutation that affect the coordination of cell division emphasizes the idea that {beta}-lactams do not kill S. aureus simply by weakening the cell wall but rather by interference with the coordination of cell division.

microbiology↗