Biology subjects

McKitterick, A. C.

Publications and source records attributed to McKitterick, A. C..

4 recordsLinked to original sources

bioRxiv2025

Bacteriophages target membrane-anchored glycopolymers to promote host cell lysis and progeny release

Most bacteriophages lyse their host cell to release progeny virions. Double-stranded DNA phages typically promote host lysis using a holin-endolysin system. Holins form pores in the cytoplasmic membrane allowing endolysins access to the peptidoglycan (PG) cell wall, which they degrade to weaken the cell envelope and promote osmotic lysis. Phages that infect Proteobacteria also encode a spanin in their lysis cassette that functions to disrupt the host outer membrane. The spanin-requirement for cell lysis provided the first clue that the proteobacterial outer membrane confers mechanical rigidity to the cell envelope. Corynebacteria and mycobacteria also build an outer membrane, but it is made of mycolic acids instead of lipopolysaccharides. Here, we investigated whether the mycomembrane presents a mechanical barrier to phage-induced lysis of corynebacteria. In addition to annotated holin and endolysin genes, mycobacteriophages and corynephages were found to encode a membrane protein we call LysZ in their lysis cassettes. Deletion of lysZ in the corynephage Cog blocked lysis of its host Corynebacterium glutamicum. Surprisingly, disruption of the host mycomembrane did not correct this phenotype. Instead, a genetic analysis revealed that blocking synthesis of membrane-anchored glycopolymers called lipomannans/lipoarabinomannans (LM/LAMs) can restore plaque formation when LysZ is inactivated. This genetic system also identified the likely flippase that transports decaprenyl-linked mannose units to the extracellular side of the membrane for polymerization into LM/LAM. Overall, our results indicate that lipoglycans like LM/LAMs play roles in mechanically stabilizing bacterial envelopes and that phages use LysZ-like factors to overcome this barrier to promote lysis. SIGNIFICANCEBacteriophages must break down the cell envelope of their host to induce cell lysis for progeny release. Pathogens like Mycobacterium tuberculosis and related corynebacteria build complex cell envelopes that are likely to be especially challenging for phages to disrupt. Here, we identify a novel lysis gene called lysZ encoded by many mycobacteriophages and corynephages. Using the model organism Corynebacterium glutamicum, we show that LysZ is required to induce host lysis. This requirement is overcome by inactivating genes needed for the synthesis of lipid-anchored glycans called lipomannans/lipoarabinomannans (LM/LAMs). These findings reveal that LM/LAMs are likely to play a mechanical role in stabilizing the cell envelope. LM/LAM biogenesis is therefore an attractive target for drugs that disrupt the integrity of mycobacterial envelopes.

microbiology↗

bioRxiv2022

Phage resistance profiling identifies new genes required for biogenesis and modification of the corynebacterial cell envelope

Bacteria of the order Corynebacteriales including pathogens such as Mycobacterium tuberculosis and Corynebacterium diphtheriae are characterized by their complex, multi-layered envelope. In addition to a peptidoglycan layer, these organisms possess an additional polysaccharide layer made of arabinogalactan and an outer membrane layer composed predominantly of long-chain fatty acids called mycolic acids. This so-called mycolata envelope structure is both a potent barrier against antibiotic entry into cells and a target of several antibacterial therapeutics. A better understanding of the mechanisms underlying mycolata envelope assembly therefore promises to reveal new ways of disrupting this unique structure for the development of antibiotics and antibiotic potentiators. Because they engage with receptors on the cell surface during infection, bacteriophages have long been used as tools to uncover important aspects of host envelope assembly. However, surprisingly little is known about the interactions between Corynebacteriales phages and their hosts. We therefore made use of the phages Cog and CL31 that infect Corynebacterium glutamicum (Cglu), a model member of the Corynebacteriales, to discover host factors important for phage infection. A high-density transposon library of Cglu was challenged with these phages followed by transposon sequencing to identify resistance loci. The analysis identified an important role for mycomembrane porins in phage infection as well as components of the arabinogalactan and mycolic acid synthesis pathways. Importantly, the approach also implicated a new gene (cgp_0396) in the process of arabinogalactan modification and identified a conserved new factor (AhfA, Cpg_0475) required for mycolic acid synthesis in Cglu.

microbiology↗

bioRxiv2020

Temporal Shifts in Antibiotic Resistance Elements Govern Virus-Pathogen Conflicts

Bacteriophage predation selects for diverse anti-phage systems that frequently cluster on mobilizable defense islands in bacterial genomes. However, there remains a lack of molecular insight into the reciprocal dynamics of phage-bacterial adaptations in nature, particularly in clinical contexts where there is need to inform phage therapy efforts and understand how phages drive pathogen evolution. Here, using time-shift experiments we show that fluctuations in SXT integrative and conjugative elements (ICEs), which notoriously confer antibiotic resistance, govern Vibrio choleraes susceptibility to phages in clinical samples. We find that SXT ICEs, which are widespread in Gammaproteobacteria, invariably encode phage defense and function to protect other genera from phage attack following conjugation. We discover phage counter-adaptation to SXT-mediated restriction in clinical samples, and show that heterogeneity in SXT ICEs allows for re-emergence of phage resistance. Further, phage infection stimulates high frequency SXT ICE conjugation, leading to the concurrent dissemination of phage and antibiotic resistance.

microbiology↗

bioRxiv2017

Anti-phage islands force their target phage to directly mediate island excision and spread

To defend against their adversaries, bacteria and phage engage in cycles of adaptation and counter-adaptation that shape their mutual evolution1-3. Vibrio cholerae, the causative agent of the diarrheal disease cholera, is antagonized by phages in the environment as well as in human hosts4,5. The lytic phage ICP1 has been recovered from cholera patient stool and water samples over at least 12 years in Bangladesh6-8 and is consequently considered a persistent predator of epidemic V. cholerae in this region. In previous work, we demonstrated that mobile genetic elements called phage-inducible chromosomal island-like elements (PLEs) protect V. cholerae from ICP1 infection7,9. PLEs initiate their anti-phage response by excising from the chromosome, however, the mechanism and molecular specificity underlying this response are not known. Here, we show that PLE 1 encodes a large serine recombinase, Int, that exploits an ICP1-specific protein, PexA, as a recombination directionality factor (RDF) to sense and excise in response to ICP1 infection. We validate the functionality and specificity of this unique recombination system, in which the recombinase and RDF are encoded in separate genomes. Additionally, we show that PexA is also hijacked to trigger excision in PLEs found in V. cholerae isolates recovered decades ago. Our results uncover an aspect of the molecular specificity underlying the longstanding conflict between a single predatory phage and V. cholerae PLE and contribute to our understanding of the molecular arms race that drives long-term evolution between combatting phage and their bacterial hosts in nature.

microbiology↗