LysX2 is a Mycobacterium tuberculosis membrane protein with an extracytoplasmic MprF-like domain

Abstract

Background Aminoacyl-phosphatidylglycerol (aaPG) synthases are bacterial enzymes that usually catalyze transfer of aminoacyl residues to the plasma membrane phospholipid phosphatidylglycerol (PG). The result is introduction of positive charges onto the cytoplasmic membrane, yielding reduced affinity towards cationic antimicrobial peptides, and increased resistance to acidic environments. Therefore, these enzymes represent an important defense mechanism for many pathogens, including Staphylococcus aureus and Mycobacterium tuberculosis (Mtb), which are known to encode for lysyl-(Lys)-PG synthase MprF and LysX, respectively. Here, we used a combination of bioinformatic, genetic and bacteriological methods to characterize a protein encoded by the Mtb genome, Rv1619, carrying a domain with high similarity to MprF-like domains, suggesting that this protein could be a new aaPG synthase family member. However, unlike homologous domains of MprF and LysX that are positioned in the cytoplasm, we predicted that the MprF-like domain in LysX2 is in the extracytoplasmic region. Results Using genetic fusions to the Escherichia coli proteins PhoA and LacZ of LysX2, we confirmed this unique membrane topology, as well as LysX and MprF as benchmarks. Expression of lysX2 in Mycobacterium smegmatis increased cell resistance to human β-defensin 2 and sodium nitrite, enhanced cell viability and delayed biofilm formation in acidic pH environment. Remarkably, MtLysX2 significantly reduced the negative charge on the bacterial surface upon exposure to an acidic environment. Additionally, we found LysX2 orthologues in major human pathogens and in rapid-growing mycobacteria frequently associated with human infections, but not in environmental and non-pathogenic mycobacteria. Conclusions Overall, our data suggest that LysX2 is a prototype of a new class within the MprF-like protein family that likely enhances survival of the pathogenic species through its catalytic domain which is exposed to the extracytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface through a yet uncharacterized mechanism.

Molecular Mechanisms of Bacterial Resistance to Antimicrobial Peptides in the Modern Era: An Updated Review

“…The deletion of lysX leads to disruptions in membrane potential, growth impairment, and defects in intracellular replication [500]. Additionally, another LysX orthologue, named LysX2, is exclusive to pathogenic mycobacteria and has been demonstrated to decrease the surface negative charge under acidic conditions, enhance bacterial cell viability at lethal pH levels, and hinder biofilm formation [502].…”

Mycobacterium tuberculosis cell-wall and antimicrobial peptides: a mission impossible?

“…Recently, Boldrin and colleagues ( 164 ) reported that lysX2 is a prototype of a new class within the MprF-like protein family that likely enhances the survival of pathogenic species through its catalytic domain, which is exposed to the extra cytoplasmic side of the cell membrane and is required to decrease the negative charge on the bacterial surface. LysX2 expression in M. smegmatis increased cell resistance to HBD-2 and sodium nitrite, improved cell viability, and delayed biofilm formation in an acidic pH environment.…”

“…The authors reported lysX2 orthologues in major human pathogens and in rapidly growing mycobacteria frequently associated with human infections, but not in environmental and nonpathogenic mycobacteria. Although the Mtb proteome presents a protein with 100% identity compared to the reported lysX2 prototype, it has not been reported whether it has the same functions as lysX ( 164 ).…”