Biology subjects

Rooke, J.

Publications and source records attributed to Rooke, J..

5 recordsLinked to original sources

bioRxiv2026

SeaB is a conserved Salmonella enterica extracellular matrix binding protein involved in biofilm formation and infection

Salmonella enterica is a leading cause of gastroenteritis worldwide. Exacerbating this issue is the emergence of multi-drug-resistant strains, posing a major threat to human health. Type 5 secretion system proteins play a major role in virulence and are viable vaccine targets. However, only a limited number of these proteins have been functionally characterized to date. In this study, we characterized SeaB, which belongs to the Type 5a secretion system. We demonstrated that SeaB is localized to the cell surface and is involved in binding to the extracellular matrix. Our results indicate that SeaB is involved in aggregation, biofilm formation and contributes to virulence. Furthermore, immunization with SeaB elicits antibodies and provides protection against Salmonella challenge in a mouse model of infection.

microbiology↗

bioRxiv2024

Identification of new genes on a whole genome scale using saturated reporter transposon mutagenesis

Small or overlapping genes are prevalent across all domains of life but are often overlooked for annotation and function because of challenges in their detection. The advent of high-density mutagenesis and data-mining studies suggest the existence of further coding potential within bacterial genomes. To overcome limitations in existing protein detection methods, we applied a genetics-based approach. We combined transposon insertion sequencing with a translation reporter to identify translated open reading frames throughout the genome at scale, independent of genome annotation. We applied our method to the well-characterised species Escherichia coli and identified [~]200 putative novel protein coding sequences (CDS). These are mostly short CDSs (<50 amino acids) and in some cases highly conserved. We validated the expression of selected CDSs demonstrating the utility of this approach. Despite the extensive study of E. coli, this method revealed proteins that have not been described previously, including proteins that are conserved and neighbour functionally important genes, suggesting significant functional roles of these small proteins. We present this as a complementary method to whole cell proteomics and ribosome trapping for condition-dependent identification of protein CDSs, and as a high-throughput method for testing conditional gene expression. We anticipate this technique will be a starting point for future high-throughput genetics investigations to determine the existence of unannotated genes in multiple bacterial species.

microbiology↗

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↗

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↗

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↗