Biology subjects

Icke, C.

Publications and source records attributed to Icke, C..

7 recordsLinked to original sources

bioRxiv2023

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multi-drug resistant clones both alarmingly associated with high mortality. The molecular microbial genetics underpinning these recalcitrant K. pneumoniae infections is unclear, coupled with the emergence of lineages resistant to nearly all present day clinically important antimicrobials. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani medium), human urine and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

microbiology↗

bioRxiv2022

TraDIS-validate: a method for curating ordered gene-replacement libraries

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterised genes. To address this gap in functional annotation, many high-throughput screens have been devised with the goal of uncovering novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered gene-replacement library. We applied our method (TraDIS-Validate) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates. TraDIS-validate therefore had a success rate of 99.7% for identifying the correct kanamycin cassette position. This dataset provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of an antibiotic resistance cassette in any ordered library.

genetics↗

bioRxiv2021

YhcB coordinates peptidoglycan and LPS biogenesis with phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all kingdoms of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the most well studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies we report the complete genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a {Delta}yhcB background. Subsequent analyses suggest that YhcB sits at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

microbiology↗

bioRxiv2020

Glycine acylation and trafficking of a new class of bacterial lipoprotein by a composite secretion system

Protein acylation is critical for many cellular functions including signal transduction, cell division and development. In bacteria, such lipoproteins have important roles in virulence and are therefore potential targets for the development of novel antimicrobials and vaccines. To date, all known bacterial lipoproteins are secreted from the cytosol via the Sec pathway, acylated on an N-terminal cysteine residue through the action of Lgt, Lsp and Lnt, and then targeted to the appropriate cellular location. In the case of Gram-negative bacteria, the lipoprotein trafficking Lol pathway transports the lipoproteins to the outer membrane where most substrate molecules are retained within the cell. Here we identify a new secretion pathway that displays the substrate lipoprotein on the cell surface. We demonstrate that the previously identified E. coli Aat secretion system is a composite system that shares similarity with type I secretion systems and elements of the Lol pathway. Remarkably, during secretion by the Aat system, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue (rather than the canonical cysteine). Mutations in AatD or CexE that disrupt glycine acylation interfere with membrane incorporation and trafficking. Our data suggest that CexE is the first member of a new class of glycine-acylated bacterial lipoprotein, while Aat represents a new secretion system that we propose be defined as a lipoprotein secretion system (LSS).

microbiology↗

bioRxiv2020

BamA and BamD are essential for the secretion of trimeric autotransporter adhesins

The BAM complex in Escherichia coli is composed of five proteins, BamA-E. BamA and BamD are essential for cell viability and are required for the assembly of {beta}-barrel outer membrane proteins. Consequently, BamA and BamD are indispensable for secretion via the classical autotransporter pathway (Type 5a secretion). In contrast, BamB, BamC and BamE are not required for the biogenesis of classical autotransporters. Recently, we demonstrated that TamA, a homologue of BamA, and its partner protein TamB, were required for efficient secretion of proteins via the classical autotransporter pathway. The trimeric autotransporters are a subset of the Type 5-secreted proteins. Unlike the classical autotransporters, they are composed of three identical polypeptide chains which must be assembled together to allow secretion of their cognate passenger domains. In contrast to the classical autotransporters, the role of the Bam and Tam complex components in the biogenesis of the trimeric autotransporters has not been investigated fully. Here, using the Salmonella enterica trimeric autotransporter SadA and the structurally similar YadA protein of Yersinia spp., we identify the importance of BamA and BamD in the biogenesis of the trimeric autotransporters and reveal that BamB, BamC, BamE, TamA and TamB are not required for secretion of functional passenger domain on the cell surface. ImportanceThe secretion of trimeric autotransporters (TAAs) has yet to be fully understood. Here we show that efficient secretion of TAAs requires the BamA and D proteins, but does not require BamB, C or E. In contrast to classical autotransporter secretion, neither trimeric autotransporter tested required TamA or B proteins to be functionally secreted.

microbiology↗

bioRxiv2020

Structure-function analyses of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation

The Gram-negative outer membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here we reveal DolP is a lipoprotein functionally conserved among Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds to anionic phospholipids through an extensive membrane:protein interface providing evidence of subcellular localization of these phospholipids within the outer membrane. This interaction is essential for DolP function and is required for sub-cellular localization of the protein to the cell division site. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.

biochemistry↗

bioRxiv2018

Mammalian Cell Entry domains are required for bile resistance and virulence in Salmonella

MCE domains were first reported in Mycobacteria as having a role in Mammalian Cell Entry, with subsequent studies showing their importance during infection. Here, we have examined the function of MCE proteins in Salmonella Typhimurium during mammalian infection. We report that MCE proteins are required for Salmonella virulence, but that this is not related to decreased adherence, entry or survival in mammalian cells. Instead, we reveal that MCE proteins are required for Salmonella bile resistance, in particular to withstand bile salts such as cholate and deoxycholate. Based on our previous work in Escherichia coli, and other studies that have reported roles for MCE proteins in membrane biogenesis, we propose that Salmonella lacking MCE domains have a defective outer membrane that results in bile sensitivity and decreased virulence in vivo. These results suggest that MCE domains mediate fundamental aspects of bacterial membrane physiology as opposed to a proposed direct role in mammalian cell entry, explaining their conservation across both pathogenic and non-pathogenic bacteria.

microbiology↗