Publications by Year: 2021

Iron is an essential element for Escherichia, Salmonella, and Shigella species. The acquisition of sufficient amounts of iron is difficult in many environments, including the intestinal tract, where these bacteria usually reside. Members of these genera have multiple iron transport systems to transport both ferrous and ferric iron. These include transporters for free ferrous iron, ferric iron associated with chelators, and heme. The numbers and types of transport systems in any species reflect the diversity of niches that it can inhabit. Many of the iron transport genes are found on mobile genetic elements or pathogenicity islands, and there is evidence of the spread of the genes among different species and pathotypes. This is notable among the pathogenic members of the genera in which iron transport systems acquired by horizontal gene transfer allow the bacteria to overcome host innate defenses that act to restrict the availability of iron to the pathogen. The need for iron is balanced by the need to avoid iron overload since excess iron is toxic to the cell. Genes for iron transport and metabolism are tightly regulated and respond to environmental cues, including iron availability, oxygen, and temperature. Master regulators, the iron sensor Fur and the Fur-regulated small RNA (sRNA) RyhB, coordinate the expression of iron transport and cellular metabolism genes in response to the availability of iron.

Iron is an essential element for Vibrio cholerae to survive, and Feo, the major bacterial system for ferrous iron transport, is important for growth of this pathogen in low-oxygen environments. To gain insight into its biochemical mechanism, we evaluated the effects of widely used ATPase inhibitors on the ATP hydrolysis activity of the N-terminal domain of V. cholerae FeoB. Our results showed that sodium orthovanadate and sodium azide effectively inhibit the catalytic activity of the N-terminal domain of V. cholerae FeoB. Further, sodium orthovanadate was the more effective inhibitor against V. cholerae ferrous iron transport in vivo. These results contribute to a more comprehensive biochemical understanding of Feo function, and shed light on designing effective inhibitors against bacterial FeoB proteins.

Staphylococcus aureus RF122 is a major pathogen that causes bovine mastitis, which is the most prevalent and costly disease in the milk and dairy industry. S. aureus expresses various virulence factors that are especially highly associated with iron metabolism, and the bacterial ferrous iron transport system Feo is important for bacterial growth or virulence in mammalian hosts. In this study, we evaluated a new antimicrobial agent, PHT-427, targeting the S. aureus RF122 Feo system for the prevention of bovine mastitis. Various analyses on in vitro enzymatic assays, growth inhibition, virulence expressions, and toxicity of animal model systems were conducted to characterize the inhibition properties of PHT-427. This small molecule efficiently inhibited enzyme activity of FeoB and bacterial growth. PHT-427 attenuated various virulence factors related to milk quality, including staphyloxanthin production, biofilm formation, and coagulation. Considering the high frequency of antibiotic-resistant S. aureus in bovine mastitis isolates, PHT-427 synergistically enhanced bacterial antibiotic susceptibility and further inhibited global Gram-positive bacterial growth. Unlike its effects on bacteria, the inhibitor did not show any toxicity on animal model systems. These results indicate that the S. aureus Feo system represents a good target for antimicrobial strategies, and this new antimicrobial agent may represent a promising biotechnological application for preventing S. aureus-induced bovine mastitis in the milk and dairy industry.

CsrA is a posttranscriptional global regulator in Vibrio cholerae Although CsrA is critical for V. cholerae survival within the mammalian host, the regulatory targets of CsrA remain mostly unknown. To identify pathways controlled by CsrA, RNA-seq transcriptome analysis was carried out by comparing the wild type and the csrA mutant grown to early exponential, mid-exponential, and stationary phases of growth. This enabled us to identify the global effects of CsrA-mediated regulation throughout the V. cholerae growth cycle. We found that CsrA regulates 22% of the V. cholerae transcriptome, with significant regulation within the gene ontology (GO) processes that involve amino acid transport and metabolism, central carbon metabolism, lipid metabolism, iron uptake, and flagellum-dependent motility. Through CsrA-RNA coimmunoprecipitation experiments, we found that CsrA binds to multiple mRNAs that encode regulatory proteins. These include transcripts encoding the major sigma factors RpoS and RpoE, which may explain how CsrA regulation affects such a large proportion of the V. cholerae transcriptome. Other direct targets include flrC, encoding a central regulator in flagellar gene expression, and aphA, encoding the virulence gene transcription factor AphA. We found that CsrA binds to the aphA mRNA both in vivo and in vitro, and CsrA significantly increases AphA protein synthesis. The increase in AphA was due to increased translation, not transcription, in the presence of CsrA, consistent with CsrA binding to the aphA transcript and enhancing its translation. CsrA is required for the virulence of V. cholerae and this study illustrates the central role of CsrA in virulence gene regulation.

The emergence of multidrug-resistant Staphylococcus aureus strains has become a serious clinical problem. Iron is absolutely required for the bacterial growth, virulence associated with colonization, and survival from the host immune system. The FeoB protein is a major iron permease in bacterial ferrous iron transport systems (Feo) that has been shown to play a crucial role in virulence of some pathogenic bacteria. However, FeoB is still uncharacterized in Gram-positive pathogens, and its effects on S. aureus pathogenesis are unknown. In this study, we identified a novel inhibitor, GW3965·HCl, that targets FeoB in S. aureus. The molecule effectively inhibited FeoB in vitro enzyme activity, bacterial growth, and virulence factor expression. Genome-editing and metabolomic analyses revealed that GW3965·HCl inhibited FeoB function and affected the associated mechanisms with reduced iron availability in S. aureus. Gentamicin resistance and Caenorhabditis elegans infection assays further demonstrated the power of GW3965·HCl as a safe and efficient antibacterial agent. In addition to S. aureus, GW3965·HCl also presented its effectiveness on inhibition of the FeoB activity and growth of Gram-positive bacteria. This novel inhibitor will provide new insight for developing a next-generation antibacterial therapy.