Jialun Wu

Listeria monocytogenes persists in food processing environments partly through its capacity to form biofilms. Yet the genetic determinants underlying variation in this trait remain poorly defined, limiting genotype-to-phenotype prediction from the whole-genome sequencing (WGS) data. We characterised the biofilm-forming ability of 25 L. monocytogenes strains isolated from clinical, food and food-processing environments and correlated the phenotype with their genome sequences. Within a cluster of closely related strains, from the same fish-processing plant, strain L1.CH4 showed a distinct biofilm phenotype. Comparative genomics identified a single-base deletion in lmo2768, producing a non-functional EslB, a membrane component of a predicted ABC transporter. Targeted disruption of eslB in closely related strains reproduced the phenotype, confirming that loss of function underlies these traits. eslB-deficient strains also formed significantly less biofilm biomass on stainless-steel coupons, with altered architecture. These phenotypes are consistent with the established role of EslB in peptidoglycan biosynthesis and cell-envelope integrity, where its loss thins the peptidoglycan layer and increases wall teichoic acid exposure. A naturally occurring, less disruptive eslB variant in another strain conferred lysozyme sensitivity without affecting biofilm formation, suggesting these functions are at least partly separate. Additionally, surveillance across public L. monocytogenes sequences revealed a high rate of premature stop codons in eslA and eslB. Altogether, these findings identify eslB as a novel genetic determinant of biofilm formation in L. monocytogenes, establishing a link between cell-envelope integrity and environmental persistence. Loss-of-function mutations in eslB may confer a selective advantage by promoting detachment and dissemination from biofilms, making eslB a useful target for genotype-based surveillance of Listeria in food processing environments.

Methicillin-resistant Staphylococcus aureus (MRSA) infections contribute significantly to the mortality rate associated with antimicrobial resistance, while also increasing medical complications when compared to methicillin-sensitive isolates. Consequently, increasing the efficacy of β-lactams may alleviate the burden associated with this drug–pathogen combination. Dialysed extracts, derived from Pelvetia canaliculata, were further partitioned to generate phlorotannin enriched extracts (PEEs). Minimum inhibitory concentrations (MICs) of extracts were used to determine the antimicrobial and antibiotic-modulating activity against seven clinical MRSA isolates. Whole-transcriptomic sequencing was carried out to determine potential mechanisms of action on the strongest extract–antibiotic combination against an MRSA isolate, determined by the fractional inhibitory concentration index (FICI). The most pronounced β-lactam modulatory effects were seen in the >100 kDa PEE from P. canaliculata in conjunction with amoxicillin (MIC fold-reductions = 12.8–214), while >30 kDa PEE resulted in the strongest FIC index value when combined with penicillin (0.23), indicating a synergistic effect. Whole-transcriptome analysis of this treatment identified multiple differentially expressed genes (Log2FC +1/−1) of statistical significance (p adj < 0.05), namely related to accelerated autolysis, cell-envelope stability, protein synthesis, DNA replication, iron homeostasis and active transport. This study highlights preliminary transcriptomic evidence of biological processes associated with penicillin modulation by P. canaliculata > 30 kDa PEE, providing a foundation for further investigation of its antibiotic-modulating activity.

In the food-borne pathogen Listeria monocytogenes , SigB is the central regulator of g eneral s tress r esponse (GSR) and it mediates host entry by promoting acid resistance and epithelial cell attachment. However, mutations can readily arise to disable r egulators of S ig B (Rsb proteins), which suggests a considerable genetic plasticity in the GSR. To further investigate this, we defined the complete genome sequence of a clinical isolate and elucidated how sequential mutations within sigB operon ( rsbX N77K and rsbU Q317*) impacted fitness through modulation of SigB activity. To investigate the plasticity of the GSR, we followed its genetic adaptation to lethal acidic challenge (mimicking the selective pressure encountered during entry into the host). Acid resistance developed rapidly and all 6 a cid r esistant d erivatives (ARDs) selected for analysis had acquired mutations in rsbW , which encodes an antagonist of SigB that suppresses SigB activity during non-stress conditions. These mutations resulted in non-canonical start codons ( rsbW ^ATG to rsbW ^ATA or rsbW ^ATT ) or premature translation termination ( rsbW ^- ) and all were found to result in increased SigB activity. A translational reporter assay demonstrated distinct differences in translation efficiency between three start codons: ATG > ATA > ATT, suggesting that a perturbation of RsbW:SigB stoichiometry alters SigB activity. We then analysed start codon usage for all conserved genes in 60,692 L. monocytogenes genomes. This analysis revealed flexible usage of start codons associated with genetic clades in 39 conserved genes, 13 of which are involved in virulence and stress response. Further, we show that flexible use of canonical start codons (ATG and GTG) can also mediate different levels of expression of virulence and stress response genes. Taken together, we show the genetic plasticity of GSR regulation in a model pathogen, and highlight the importance of translational control as a means of fine-tuning gene expression during short-term adaptation and long-term evolution for optimal fitness.

Under acidic conditions, the glutamate decarboxylase system GAD2 protects Listeria monocytogenes by consuming intracellular protons. GAD2 is encoded by the gadT2D2 operon and regulated by the transcriptional regulator GadR. In this study we investigated whether osmotic stress, which is commonly used as a hurdle in food preservation, influences GadR activity. Exposure to 0.5 M or 1 M NaCl rapidly increased gadT2 transcription in a GadR-dependent manner. This induction was independent of Na+ or Cl- ions as other osmotically active molecules (e.g. KCl, Na2SO4, sucrose) produced similar effects, suggesting that this regulation is responsive to changes in osmolarity. Two putative GadR binding sites in PgadT2 were indispensable for this osmotic induction. Furthermore, GadR-dependent GAD2 expression is elevated in NaCl-treated cultures and it confers protection against lethal acid stress. Taken together these data showed that osmotic stress induces GadR-dependent GAD2 expression, which confers a significant survival advantage when cells are subsequently exposed to acid stress, likely due to an improved capacity for intracellular pH homeostasis. Thus, GadR is activated by two conditions commonly used in food preservation, mild acid pH and elevated osmolarity, which may increase bacterial resistance to gastric acid, posing a potential risk to food safety.

Introduction. Molecular epidemiological and phenotypic analyses of Listeria monocytogenes strains can inform our understanding of factors that influence onward transmission, virulence potential and ability to control the pathogen in foods or in clinical settings. Bile acids represent a host-specific barrier to microbial colonization of the gastrointestinal tract and are, therefore, not encountered in the external environment. We tested the hypothesis that tolerance of bile acids may be an evolutionary adaptation across L. monocytogenes clonal complexes (CCs), which varies with genotype and/or is associated with clinical isolates. Hypothesis. We hypothesized that strains of L. monocytogenes may differ in bile tolerance (a potential virulence-associated trait) and herein examine this phenomenon according to CC, strain origin and genotype. Aim. To assess 205 genome-sequenced isolates of L. monocytogenes for tolerance of porcine bile acids and bile salt hydrolase (BSH) activity. Methodology. Survival of L. monocytogenes strains was determined following exposure to porcine bile acids under conditions that mimic the small intestinal environment. BSH activity was assayed against pure taurodeoxycholic acid and glycodeoxycholic acid using an agar plate deconjugation assay. Genomes were analysed for polymorphisms in known bile tolerance loci. Results. Isolates demonstrated distinct inter-individual variances in bile tolerance under anaerobic conditions that mimic the intestinal environment. Strains isolated from cases of human disease were significantly more bile-tolerant than those isolated from natural environments or foods. There was no correlation between levels of bile tolerance and the size of bile precipitation zones on the BSH agar plate assay. No significant patterns were seen upon analysis of known or putative bile tolerance loci; however, individual strains with naturally occurring sigB operon mutations demonstrated reduced bile tolerance. Conclusion. L. monocytogenes strains isolated from clinical cases of listeriosis demonstrated elevated bile resistance consistent with a likely enhanced capacity to cause gastrointestinal infection preceding invasive disease. The data suggest the potential importance of bile tolerance in L. monocytogenes infection and highlight underlying molecular mechanisms by which strains vary in their natural levels of bile tolerance.

In the food-borne pathogen Listeria monocytogenes , SigB is the central regulator of general stress response (GSR) and it mediates host entry by promoting acid resistance and epithelial cell attachment. The post-translational regulation of SigB activity is complex and is controlled by multiple genes. There is significant evidence that mutations can readily arise in these regulatory genes, or in sigB itself, leading to reduced SigB activity, which suggests that there is considerable genetic plasticity In the GSR. To further investigate this, we defined the complete genome sequence of a clinical isolate with attenuated SigB activity and investigated how it adapts to lethal acidic challenge (mimicking the selective pressure encountered during entry into the host). Acid resistance developed rapidly and 6 acid resistant derivatives (ARDs) were selected for further investigation. All 6 ARDs restored SigB activity due to mutations acquired in rsbW , which encodes an antagonist of SigB. These mutations resulted in non-canonical start codons ( rsbW ^ATG to rsbW ^ATA or rsbW ^ATT ) or premature translation termination ( rsbW ^- ). A translational reporter assay demonstrated distinct differences in translation efficiency between three start codons: ATG>ATA>ATT, suggesting that a perturbation of RsbW:SigB stoichiometry alters SigB activity. We then analysed start codon usage for all conserved genes in 60,692 L. monocytogenes genomes available in the NCBI database. This analysis revealed flexible usage of start codons associated with genetic clades in 39 conserved genes, 13 of which are involved in virulence and stress response. Further, we show that flexible use of canonical start codons (ATG and GTG) also mediates different levels of expression of virulence and stress response genes. Taken together, we show the genetic plasticity of GSR regulation in a model pathogen, and highlight the importance of translational control as a means of fine-tuning gene expression during short-term adaptation and long-term evolution for optimal fitness. Author Summary: The general stress response (GSR) in foodborne pathogen Listeria monocytogenes is important for environmental stress response and for host entry, but GSR is also highly variable across wild isolates. In this study, we analysed the evolutionary trajectory of a clinical isolate with attenuated GSR and characterized the adaption by this strain to a host-mimicking stress (acidic condition). Under extreme selective pressure, mutations disabling a negative regulator of the GSR were enriched. Interestingly, several independently occurring mutations negatively affect the translation initiation by using non-canonical start codons. Prompted by this, we analysed the population-wide start codon usage by examining all available L. monocytogenes genomes available (n = 60,690). This analysis revealed differential start codon usage in 39 conserved genes that are associated with different genetic clades. Furthermore, we demonstrated differential translation efficiencies between the different canonical start codons. This work highlights the genetic plasticity of GSR in the important food-borne pathogen L. monocytogenes and shows that altered translational start codon can be used as means of regulatory control. Together the data suggest that genetic changes in the regulation of the GSR might confer niche-specific fitness advantages.

The ability to sense and respond effectively to acidic stress is important for microorganisms to survive and proliferate in fluctuating environments. As specific metabolic activities can serve to buffer the cytoplasmic pH, microorganisms re-wire their metabolism to favour these reactions and thereby mitigate acid stress. The orally-acquired pathogen Listeria monocytogenes exploits alternative metabolic activities to overcome the acidic stress encountered in the human stomach or food products. In this minireview, we discuss the metabolic processes in L. monocytogenes that mitigate acid stress, with an emphasis on the proton-depleting reactions including glutamate decarboxylation, arginine/agmatine deimination, and fermentative acetoin production. We also summarize the recent findings on regulatory mechanisms that control the expression of genes that are responsible for these metabolic activities, including the general stress response regulator SigB, arginine repressor ArgR, and the recently discovered RofA-like transcriptional regulatory GadR. We further discuss the importance of this metabolic reprogramming in the context of food products and within the host. Finally, we highlight some outstanding challenges in the field including an understanding of acid-sensing mechanisms, the role of intra-species heterogeneity in acid resistance, and how a fundamental understanding of acid stress response can be exploited for food formulation to improve food safety and reduce food waste.

Stomach acid provides a significant innate barrier to the entry of the food-borne pathogen Listeria monocytogenes into the human gastrointestinal tract. A key determinant of acid resistance in this bacterium is the conserved glutamate decarboxylase system, GadD2 (encoded by the gadT2D2 operon), which helps to maintain the intracellular pH during exposure to gastric acid. In this study, we identified a premature stop codon in a gene located immediately downstream of the gadT2D2 operon that was highly linked to an acid-sensitive phenotype. When this open reading frame was restored through homologous recombination, an acid-resistant phenotype was restored. Through a series of genetic, transcriptomic, and survival experiments, we established that this gene, which we designated gadR , encodes a transcriptional regulator of the gadT2D2 operon. GadR belongs to the RofA family of regulators, primarily found in streptococci, where they are involved in regulating virulence. The data further showed that gadR plays a critical role in the development of acid resistance in response to mild acid exposure, a response that is known as the adaptive acid tolerance response (ATR). A deletion analysis of the gadT2D2 promoter region identified two 18-bp palindromic sequences that are required for the GadR-mediated induction of gadT2D2 , suggesting that they act as binding sites for GadR. Overall, this study uncovers a new RofA-like regulator of acid resistance in L. monocytogenes , which plays a significant role in both growth phase-dependent acid resistance and ATR and accounts for previously observed strain-to-strain differences in survival at low pH. IMPORTANCE: The ability to survive the acidic conditions found in the stomach is crucial for the food-borne pathogen Listeria monocytogenes to gain access to the mammalian gastrointestinal tract. Little is currently known about how acid resistance is regulated in this pathogen and why this trait is highly variable between strains. Here, we used comparative genomics to identify a novel RofA-family transcriptional regulator, GadR, that controls the development of acid resistance. The RofA family of regulators was previously found only in a small group of bacterial pathogens, including streptococci, where they regulate virulence properties. We show that gadR encodes the dominant regulator of acid resistance in L. monocytogenes and that its sequence variability accounts for previously observed differences between strains in this trait. Together, these findings significantly advance our understanding of how this important pathogen copes with acid stress and suggest a potential molecular target to aid its control in the food chain.

Stomach acid provides a significant innate barrier to the entry of the food-borne pathogen Listeria monocytogenes into the human gastrointestinal tract. A key determinant of acid resistance in this bacterium is the conserved glutamate decarboxylase system, GadD2 (encoded by the gadT2D2 operon), which helps to maintain the intracellular pH during exposure to gastric acid. In this study, we identified a premature stop codon in a gene located immediately downstream of the gadT2D2 operon that was highly linked to an acid sensitive phenotype. When this open reading frame was restored through homologous recombination an acid resistant phenotype resulted. Through a series of genetic, transcriptomic and survival experiments we established that this gene, which we designated gadR , encodes a transcriptional regulator of the gadT2D2 operon. GadR belongs to the RofA family of regulators, primarily found in the streptococci, where they are involved in regulating virulence. The data further showed that gadR plays a critical role in the development of acid resistance in response to mild acid exposure, a response that is known as the adaptive acid tolerance response (ATR). A deletion analysis of the gadT2D2 promoter region identified two 18bp palindromic sequences that are required for the GadR-mediated induction of gadT2D2 , suggesting that they act as binding sites for GadR. Overall, this study uncovers a new RofA-like regulator of acid resistance in L. monocytogenes that plays a significant role in both growth phase-dependent and ATR mediated acid resistance and accounts for previously observed strain-to-strain differences in survival at low pH. The findings have important implications for understanding the behavior of L. monocytogenes in acidic environments and identify a potential target for improved control of this important pathogen. Author summary: The ability to survive the acidic conditions found in the stomach is a key trait enabling the food-borne pathogen Listeria monocytogenes to gain access to mammalian gastrointestinal tract, where it can initiate an infection. Little is currently known about how acid resistance is regulated in this pathogen and why this trait is highly variable between strains. Here we used genomic sequences from a collection of L. monocytogenes strains with known differences in acid survival to identify a novel transcriptional regulator controlling acid resistance, which we call GadR. The regulator belongs to a family of regulators previously found only in a small group of bacterial pathogens including the streptococci, where they are involved in regulating virulence properties. We show that GadR is the dominant regulator of acid resistance in L. monocytogenes and that variability in its gene sequence accounts for previously observed differences between strains in this trait. Together these findings significantly advance our understanding of how this important pathogen copes with acid stress and suggests a potential molecular target to better control it in the human food-chain.

The bacterial foodborne pathogen Listeria monocytogenes frequently contaminates various categories of food products and is able to cause life-threatening infections when ingested by humans. Thus, it is important to control the growth of this bacterium in food by understanding the mechanisms that allow its proliferation under suboptimal conditions.