Biology subjects

Moynihan, P. J.

Publications and source records attributed to Moynihan, P. J..

6 recordsLinked to original sources

bioRxiv2026

Host Cell Wall Polysaccharides Modulate Nanosynbacteria Recognition and Attachment

Ultrasmall episymbiotic bacteria grow on the surface of their obligate bacterial hosts. These episymbionts have a restricted host range and likely rely on host-derived receptor molecules to identify and associate with cognate hosts. Nanosynbacteria from the oral microbiome, like Nanosynbacter lyticus, utilize a pair of distinct type 4 pili (T4P) for host surface interactions, T4P-1 and T4P-2. In this study we identify the adhesin components of both T4P, demonstrate that these adhesins localize to the cell wall of the cognate host Actinobacterium Schaalia odontolytica, and demonstrate that each adhesin binds distinct components of the host cell wall. The adhesin from the motility driving T4P-1, termed PilX1, showed a wide range of affinities in microarray screening and bound peptidoglycan directly, but could not bind purified cell wall polysaccharides (CWPS) from host surfaces. Conversely, the adhesin from T4P-2, termed PilX2, bound host-derived CWPS and preferentially bound episymbiont-naive hosts over episymbiont-exposed hosts. Within the bacterial host we identified two CWPS biosynthesis pathways that altered adhesin binding and episymbiont susceptibility. Disruption of a rhamnose-CWPS pathway left hosts susceptible to episymbiont binding and disruption of a fucose remodeling pathway made hosts more resistant to episymbiont binding. Analyzing polysaccharide compositions revealed multiple CWPS, potentially including a rhamnose- and mannose-rich rhamnomannan. Consistent with this finding, exogenously added rhamnose and mannose were able to block binding by N. lyticus, supporting a role for these sugars in episymbiont defense. Our study reveals that Nanosynbacteria rely on the complex sugars that coat their host bacteria for recognition and attachment.

microbiology↗

bioRxiv2026

Functional Differentiation of GH172 Arabinofuranosidases Through Divergent Quaternary Structures

Mycobacteria synthesise the unusual glycan [x1D05]-arabinan as a major component of the cell wall glycoconjugates arabinogalactan (AG) and lipoarabinomannan (LAM). We previously identified Dysgonomonas gadei, a member of the Bacteroidota, as capable of complete [x1D05]-arabinan degradation through the concerted action of endo- and exo-acting enzymes. Among these are three glycoside hydrolase family 172 (GH172) enzymes with exo--[x1D05]-arabinofuranosidase activity against AG and LAM, although their linkage specificities were unknown. Here, using defined synthetic substrates, we show that the three enzymes possess distinct linkage preferences. We also develop -[x1D05]-arabinofuranosyl cyclophellitol aziridines as covalent inhibitors and activity-based probes for GH172 enzymes. X-ray crystallography and cryo-EM to reveal strikingly different quaternary assemblies across the three homologues, while a 1.5 [A] cryo-EM structure of dodecameric Dg67 covalently modified by an aziridine inhibitor identifies the catalytic nucleophile and provides direct structural support for a retaining mechanism. A BODIPY-tagged aziridine probe selectively labelled the three GH172 enzymes in D. gadei cell lysates. Together, these findings define functional and structural diversity within GH172 and establish chemical probes for profiling -[x1D05]-arabinofuranosidase activity in complex biological samples.

biochemistry↗

bioRxiv2026

Direct targeting of the WAVE-regulatory complex and the actin cytoskeleton by the Anaplasma phagocytophilum effector, AnkA

The obligate intracellular tick-borne pathogen Anaplasma phagocytophilum is unusual in its tropism for neutrophils, within which it survives and replicates. Central to the ability of this bacterium to colonize this hostile niche are secreted effector proteins. One such effector protein, AnkA, interacts with multiple host ligands in both the cytoplasm and the nucleus, yet how these interactions promote infection is largely unknown. We discovered that AnkA directly binds both the WAVE Regulatory Complex (WRC), a signal-integration hub that regulates the formation of branched actin networks underpinning diverse cell functions, and to monomeric and filamentous actin. We determined that AnkA uses two forms of molecular mimicry, first by binding to the Abi1 component of the WRC via a WRC-Recruiting Abi-binding Peptide (WRAP), and second by binding to actin via a WH2-like Actin Binding Motif (ABM). Via these interactions, AnkA can exert multi-factorial influence on the actin cytoskeleton and signaling pathways converging on this host structure. AnkA and the WRC co-localise during bacterial internalization and during the late stages of infection, suggesting a central role in both host-cell colonization and bacterial dissemination. Our work reveals AnkA as the first example of a bacterial effector that directly targets the WRC. The co-occurrence of WRAP- and ABM-like motifs in other bacterial effector proteins suggests that this mechanism of actin cytoskeleton subversion may represent a conserved strategy used by other obligate intracellular bacteria.

microbiology↗

bioRxiv2023

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important amongst these are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are both trafficked out of the bacterium to the host via unknown mechanisms. An important class of exported LM/LAM is the capsular derivative of these molecules which is devoid of its lipid anchor. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH which specifically cleaves -1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures where arabinomannan acts as a signal for growth phase transition. Finally, we demonstrate that LamH is important for Mycobacterium tuberculosis survival in macrophages. These data provide a new framework for understanding the biological role of LAM in mycobacteria.

microbiology↗

bioRxiv2022

Mining the human gut microbiome identifies mycobacterial D-arabinan degrading enzymes

Division and degradation of bacterial cell walls requires coordinated action of a myriad of enzymes. This particularly applies to the elaborate cell walls of acid-fast organisms such as Mycobacterium tuberculosis, which consist of a multi-layered cell wall that contains an unusual glycan called arabinogalactan. Enzymes that cleave the O_SCPLOWDC_SCPLOW-arabinan core of this structure have not previously been identified in any organism. We have interrogated the diverse carbohydrate degrading enzymes expressed by the human gut microbiota and uncovered four families of glycoside hydrolases with the capability to degrade the O_SCPLOWDC_SCPLOW-arabinan or O_SCPLOWDC_SCPLOW-galactan components of arabinogalactan. Using novel exo-O_SCPLOWDC_SCPLOW-galactofuranosidases from gut bacteria we generated enriched O_SCPLOWDC_SCPLOW-arabinan and used it to identify D. gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan. We have identified new members of the DUF2961 family (GH172), and a novel family of glycoside hydrolases (DUF4185) that display endo-O_SCPLOWDC_SCPLOW-arabinofuranase activity. The DUF4185 enzymes are conserved in mycobacteria and found in many microbes, suggesting that the ability to degrade mycobacterial glycans plays an important role in the biology of diverse organisms. All mycobacteria encode two conserved endo-O_SCPLOWDC_SCPLOW-arabinanases that display different preferences for the O_SCPLOWDC_SCPLOW-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.

biochemistry↗

bioRxiv2018

Mycobacteria recycle their peptidoglycan via a novel pathway which influences antimicrobial resistance and limits proliferation in macrophages.

Growth and division by most bacteria requires remodeling and cleavage of their cell wall. A byproduct of this process is the generation of free peptidoglycan (PG) fragments known as muropeptides. These muropeptides are recycled in many model organisms, where the bacteria can harness their unique nature as a signal for cell wall damage. These molecules also serve as important signals for hosts where binding to specific receptors reports on the presence of intracellular bacteria. Despite this critical role for muropeptides, it has long been thought that pathogenic mycobacteria such as Mycobacterium tuberculosis do not recycle their PG. Herein we show for the first time that M. tuberculosis and Mycobacterium bovis BCG are able to recycle components of their PG. We demonstrate that the core-mycobacterial gene lpqI, encodes an authentic NagZ {beta}-N-acetylglucosaminidase and that it is essential for PG-derived amino sugar recycling via an unusual pathway. By characterizing an M. bovis BCG strain lacking lpqI we are also able to show that stem-peptide recycling proceeds independent of amino sugar recovery and loss of lpqI leads to antimicrobial resistance in vitro. Together these data provide a critical first step in understanding how mycobacteria recycle their peptidoglycan.

microbiology↗