Identification of a Novel Regulator of Clostridioides difficile Cortex Formation.

Clostridioides difficile is a leading cause of health care-associated infections worldwide. These infections are transmitted by C. difficile's metabolically dormant, aerotolerant spore form. Functional spore formation depends on the assembly of two protective layers, a thick layer of modified peptidoglycan known as the cortex layer and a multilayered proteinaceous meshwork known as the coat. We previously identified two spore morphogenetic proteins, SpoIVA and SipL, that are essential for recruiting coat proteins to the developing forespore and making functional spores. While SpoIVA and SipL directly interact, the identities of the proteins they recruit to the forespore remained unknown. Here, we used mass spectrometry-based affinity proteomics to identify proteins that interact with the SpoIVA-SipL complex. These analyses identified the Peptostreptococcaceae family-specific, sporulation-induced bitopic membrane protein CD3457 (renamed SpoVQ) as a protein that interacts with SipL and SpoIVA. Loss of SpoVQ decreased heat-resistant spore formation by ∼5-fold and reduced cortex thickness ∼2-fold; the thinner cortex layer of ΔspoVQ spores correlated with higher levels of spontaneous germination (i.e., in the absence of germinant). Notably, loss of SpoVQ in either spoIVA or sipL mutants prevented cortex synthesis altogether and greatly impaired the localization of a SipL-mCherry fusion protein around the forespore. Thus, SpoVQ is a novel regulator of C. difficile cortex synthesis that appears to link cortex and coat formation. The identification of SpoVQ as a spore morphogenetic protein further highlights how Peptostreptococcaceae family-specific mechanisms control spore formation in C. difficile. IMPORTANCE The Centers for Disease Control has designated Clostridioides difficile as an urgent threat because of its intrinsic antibiotic resistance. C. difficile persists in the presence of antibiotics in part because it makes metabolically dormant spores. While recent work has shown that preventing the formation of infectious spores can reduce C. difficile disease recurrence, more selective antisporulation therapies are needed. The identification of spore morphogenetic factors specific to C. difficile would facilitate the development of such therapies. In this study, we identified SpoVQ (CD3457) as a spore morphogenetic protein specific to the Peptostreptococcaceae family that regulates the formation of C. difficile's protective spore cortex layer. SpoVQ acts in concert with the known spore coat morphogenetic factors, SpoIVA and SipL, to link formation of the protective coat and cortex layers. These data reveal a novel pathway that could be targeted to prevent the formation of infectious C. difficile spores.

SpoIVA-SipL Complex Formation Is Essential for Clostridioides difficile Spore Assembly.

Spores are the major infectious particle of the Gram-positive nosocomial pathogen Clostridioides difficile (formerly Clostridium difficile), but the molecular details of how this organism forms these metabolically dormant cells remain poorly characterized. The composition of the spore coat in C. difficile differs markedly from that defined in the well-studied organism Bacillus subtilis, with only 25% of the ∼70 spore coat proteins being conserved between the two organisms and with only 2 of 9 coat assembly (morphogenetic) proteins defined in B. subtilis having homologs in C. difficile We previously identified SipL as a clostridium-specific coat protein essential for functional spore formation. Heterologous expression analyses in Escherichia coli revealed that SipL directly interacts with C. difficile SpoIVA, a coat-morphogenetic protein conserved in all spore-forming organisms, through SipL's C-terminal LysM domain. In this study, we show that SpoIVA-SipL binding is essential for C. difficile spore formation and identify specific residues within the LysM domain that stabilize this interaction. Fluorescence microscopy analyses indicate that binding of SipL's LysM domain to SpoIVA is required for SipL to localize to the forespore while SpoIVA requires SipL to promote encasement of SpoIVA around the forespore. Since we also show that clostridial LysM domains are functionally interchangeable at least in C. difficile, the basic mechanism for SipL-dependent assembly of clostridial spore coats may be conserved.IMPORTANCE The metabolically dormant spore form of the major nosocomial pathogen Clostridioides difficile is its major infectious particle. However, the mechanisms controlling the formation of this resistant cell type are not well understood, particularly with respect to its outermost layer, the spore coat. We previously identified two spore-morphogenetic proteins in C. difficile: SpoIVA, which is conserved in all spore-forming organisms, and SipL, which is conserved only in the clostridia. Both SpoIVA and SipL are essential for heat-resistant spore formation and directly interact through SipL's C-terminal LysM domain. In this study, we demonstrate that the LysM domain is critical for SipL and SpoIVA function, likely by helping recruit SipL to the forespore during spore morphogenesis. We further identified residues within the LysM domain that are important for binding SpoIVA and, thus, functional spore formation. These findings provide important insight into the molecular mechanisms controlling the assembly of infectious C. difficile spores.

A Screen for Protein-Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A.

Outer membrane lipids in pathogenic mycobacteria are important for virulence and survival. Although the biosynthesis of these lipids has been extensively studied, mechanisms responsible for their assembly in the outer membrane are not understood. In the study of Gram-negative outer membrane assembly, protein-protein interactions define transport mechanisms, but analogous interactions have not been explored in mycobacteria. Here we identified interactions with the lipid transport protein LprG. Using site-specific photo-cross-linking in live mycobacteria, we mapped three major interaction interfaces within LprG. We went on to identify proteins that cross-link at the entrance to the lipid binding pocket, an area likely relevant to LprG transport function. We verified LprG site-specific interactions with two hits, the conserved lipoproteins LppK and LppI. We further showed that LprG interacts physically and functionally with the mycolyltransferase Ag85A, as loss of either protein leads to similar defects in cell growth and mycolylation. Overall, our results support a model in which protein interactions coordinate multiple pathways in outer membrane biogenesis and connect lipid biosynthesis to transport.

Transport of outer membrane lipids in mycobacteria.

The complex organization of the mycobacterial cell wall poses unique challenges for the study of its assembly. Although mycobacteria are classified evolutionarily as Gram-positive bacteria, their cell wall architecture more closely resembles that of Gram-negative organisms. They possess not only an inner cytoplasmic membrane, but also a bilayer outer membrane that encloses an aqueous periplasm and includes diverse lipids that are required for the survival and virulence of pathogenic species. Questions surrounding how mycobacterial outer membrane lipids are transported from where they are made in the cytoplasm to where they function at the cell exterior are thus similar, and similarly compelling, to those that have driven the study of Gram-negative outer membrane transport pathways. However, little is understood about these processes in mycobacteria. Here we contextualize these questions by comparing our current knowledge of mycobacteria with better-defined systems in other organisms. Based on this analysis, we propose possible models and highlight continuing challenges to improving our understanding of outer membrane assembly in these medically and environmentally important bacteria. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.

Diacyltransferase Activity and Chain Length Specificity of Mycobacterium tuberculosis PapA5 in the Synthesis of Alkyl ß-Diol Lipids.

Although they are classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl ß-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. Here, we show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl ß-diol substrate analogues. By applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and that a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in the regulation of DIM biosynthesis.

The rv1184c locus encodes Chp2, an acyltransferase in Mycobacterium tuberculosis polyacyltrehalose lipid biosynthesis.

Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption of chp2 led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment of M. tuberculosis resulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to produce in vitro reconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates.