Mycobacterium tuberculosis CwsA Interacts with CrgA and Wag31, and the CrgA-CwsA Complex Is Involved in Peptidoglycan Synthesis and Cell Shape Determination

Abstract

cBacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein from Mycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog in M. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [ 14 C]N-acetylglucosamine cell wall precursor. The M. smegmatis MSMEG_0023 crgA double mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA for cell wall synthesis and cell shape protein A, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria. Since its reemergence in the early 1990s, tuberculosis caused by Mycobacterium tuberculosis remains a leading cause of global morbidity and mortality. With nearly 10 million new cases reported each year and 2 billion people infected, as well as the emergence of extensively drug-resistant and completely drug-resistant strains, sustained interest in improved control measures and tuberculosis research is crucial (4, 40). These reports emphasize the importance of understanding the basic biology of the pathways needed for pathogen proliferation, namely, DNA replication and cell division, and the development of novel strategies for combating M. tuberculosis infection (27).Cell division in bacteria is accomplished by the coordinated actions of multiple proteins that form a septal ring in the middle of a dividing cell. FtsZ, a cytoskeletal protein and a GTPase, initiates the cytokinesis process by the GTP-dependent midcell assembly of a ring structure called the Z ring (2). Constriction of the Z ring, driven by the GTPase activity of FtsZ, initiates cell division. During various stages of cell division, FtsZ interacts with a number of proteins, and these interactions modulate its midcell localization, membrane tethering, biochemical activities, and influence on the assembly of other septal ring members, including some proteins involved in peptidoglycan (PG) synthesis (reviewed in reference 2). Recent data also suggest that, at least in some bacteria, FtsZ plays a role in guiding cell wall synthesis (1, 37).In the last decade, significant advancements have been made not only ...

Structure of CrgA, a cell division structural and regulatory protein from Mycobacterium tuberculosis , in lipid bilayers

“…This sequence has a plethora of surfaces for protein interactions to the extent that large hydrophobic residues that provide the majority of the van der Waals interactions for helix-helix binding are scarce. A cwsA and crgA double-deletion mutant showed that these proteins are required for cell wall PG synthesis and cell morphology (5). Fig.…”

“…Moreover, CrgA appears to have an important role in peptidoglycan (PG) formation during cell division, by recruiting PG synthases to the divisome (4). Reduced production of CrgA results in elongated cells and reduced growth rate (1), and loss of CrgA impairs PG synthesis (5). In addition to CwsA, the Mtb divisome involves other atypical players such as FipA (FhaB), ChiZ, and MtrB (6-8), and thus there is much yet to be learned about the participants in mycobacterial cell division (9).…”

Mycobacterium tuberculosis MtrB Sensor Kinase Interactions with FtsI and Wag31 Proteins Reveal a Role for MtrB Distinct from That Regulating MtrA Activities

“…In addition to FtsZ and the MtrB proteins, previous studies have shown that FtsI, FtsW, FipA, Wag31, CrgA, CwsA, RipA, and penicillin-binding protein A (PBPA) are other necessary septasomal components for the cell division in mycobacteria (6,(8)(9)(10)(11)(12)(13)(14)(15)(16). These studies also established pairwise interactions between FtsI and Wag31, FtsI and FtsW, FtsI and CrgA, CwsA and Wag31, and CrgA and PBPA.…”

“…Wag31 localizes in the WT cells primarily to the cell poles, with ϳ7% of cells showing a midcell localization (6,12) (Fig. 4A, panel ii).…”

“…Published data indicate that in mycobacteria, as in other Gram-positive bacteria, not only the known cell division proteins, such as FtsZ, FtsI, and Wag31, but also those of the sensor kinases belonging to the TCRS and STPK system, e.g., MtrB and PknA/B, and novel small membrane proteins, e.g., CwsA and CrgA, localize at the septa (6,8,10,12,14,27). Our data take these observations to the next level, define interactions among select septasomal components, namely, MtrB with FtsI and Wag31, and evaluate the consequences of these interactions.…”