Biology subjects

Oglesby, A. G.

Publications and source records attributed to Oglesby, A. G..

7 recordsLinked to original sources

bioRxiv2026

RsmW is an iron-responsive 3 UTR derived small regulatory RNA that contributes to biofilm formation and virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes acute and chronic opportunistic infections. Progression to chronic infection requires upregulation of the Rsm small regulatory RNAs (sRNAs), which in turn induce the expression of virulence factors to promote chronic virulence phenotypes, including biofilm formation. Iron is a critical nutrient for survival and virulence of Pa, and host-mediated iron limitation leads to the induction of key virulence factors driving infection. Prior work showed that the RsmY and RsmZ sRNAs are modestly upregulated due to iron limitation in static growth conditions via PqsR. Here, we show that another Rsm sRNA, RsmW, is strongly induced by iron limitation, likely in a Fur-dependent manner. We identified an RNA processing event, which appears to be mediated by the RNAse E, resulting in cleavage of RsmW from the 3 UTR of pa4570 mRNA. We also report that RsmW is more stable than the full length pa4570-rsmW transcript as well as the processed pa4570 mRNA. Surprisingly, we found that RetS, which down-regulates RsmY and RsmZ expression, enhances expression of RsmW under iron limiting conditions. We further demonstrate that RsmW plays a key role in biofilm formation under iron limiting conditions and is required for full pathogenesis in a Galleria mellonella infection model. Lastly, we observed similar iron-dependent regulation of RsmW in chronic infection isolates from cystic fibrosis (CF) sputum, underlining the potential importance of this regulation in the CF lung. Taken together, the results outline a new paradigm for sRNA-dependent iron regulation of P. aeruginosa pathogenesis.

microbiology↗

bioRxiv2025

Iron and peroxide regulation of the PrrF sRNAs and a conserved Dps-like protein in Pseudomonas aeruginosa and Pseudomonas fluorescens

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron to cause infection. Iron can also be toxic due to its participation in Fenton chemistry, resulting in the production of reactive oxygen species (ROS). Thus, P. aeruginosa regulates the uptake, use, and storage of iron to mitigate the effects of ROS. P. aeruginosa uses several mechanisms to manage oxidative stress, including superoxide dismutases to detoxify the superoxide radical, catalases to break down H2O2, and members of the ferritin superfamily to store iron. The iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs) are predicted to pair with and destabilize mRNA transcripts for several oxidative stress response proteins, including sodB, katA, and brnD, encoding a novel bacterioferritin-like Dps protein. In this study, we developed brnD reporter constructs that are responsive to PrrF-mediated iron regulation. We demonstrated that PrrF-mediated regulation of brnD occurs in the 5 untranslated region (UTR) of its mRNA, likely via a conserved region of complementarity with the PrrF sRNAs. We further demonstrated that brnD mRNA levels are increased upon hydrogen peroxide treatment. Surprisingly, peroxide treatment also resulted in elevated levels of the PrrF sRNAs, and this induction occurred specifically at the prrF2 promoter. Lastly, we investigated these regulatory effects in Pseudomonas fluorescens, revealing similar iron regulation of PrrF sRNAs and the brnD ortholog, as well as peroxide-induced expression of the PrrF sRNAs. Combined, these data highlight a distinct class of iron-responsive Dps-like proteins with potential functional conservation across the pseudomonads, and they reveal a novel aspect of the oxidative stress response involving the PrrF sRNAs. IMPORTANCEIron is a crucial micronutrient for Pseudomonas aeruginosa survival and virulence, yet it can also be toxic due to production of reactive oxygen species. The PrrF sRNAs are produced in iron limiting conditions and block the expression of several mRNAs involved in P. aeruginosas oxidative stress response. Here, we make the surprising discovery that oxidative stress induces expression of the PrrF sRNAs. We also provide evidence that PrrF regulation of two mRNA targets is hindered upon oxidative stress. Oxidative stress similarly induced the PrrF sRNAs in Pseudomonas fluorescens, indicating conservation of this novel regulatory event. This study therefore highlights a novel and conserved regulatory link between iron homeostasis and oxidative stress protection in the pseudomonads.

microbiology↗

bioRxiv2025

Calprotectin protects Staphylococcus aureus in coculture with Pseudomonas aeruginosa by attenuating quorum sensing and decreasing the production of pseudomonal antimicrobials

Pseudomonas aeruginosa and Staphylococcus aureus cause debilitating polymicrobial infections in diverse patient populations. Studies of these bacterial pathogens in coculture have shown that environmental variables including Fe availability and the host-defense protein calprotectin (CP) impact coculture dynamics. To decipher how CP modulates interactions between P. aeruginosa and S. aureus, we employed dual-species RNA-seq to examine the transcriptional responses of both pathogens in coculture to CP treatment and metal depletion. Analysis of these responses revealed that, for both P. aeruginosa and S. aureus, CP treatment not only induced gene expression changes consistent with single- and multi-metal starvation responses, but also induced gene expression changes that were not observed under metal limitation. For P. aeruginosa, CP treatment induced gene expression changes pointing to a shift in chorismate flux away from alkylquinolone and phenazine biosynthesis towards folate biosynthesis. These observations were consistent with decreased production of alkylquinolones by P. aeruginosa, including the potent anti-staphylococcal alkylquinolone N-oxides. CP treatment afforded perturbed levels of two quorum sensing molecules, 3-oxo-C12-homoserine lactone and C4-homoserine lactone, produced by P. aeruginosa. In addition, CP treatment enhanced the ability of S. aureus to mount Fe starvation responses and caused S. aureus to express host virulence genes. This analysis illuminated physiological consequences of CP treatment that extend beyond metal starvation and that these consequences impact interspecies interactions. Our findings provide a working model in which CP effectively disarms P. aeruginosa by inhibiting the production of anti-staphylococcal factors and boosts the ability of S. aureus to protect itself from attack. ImportanceThe innate immune protein calprotectin (CP) defends the host against bacterial pathogens by sequestering multiple essential nutrient metal ions at infection sites. In addition to this role in nutritional immunity, CP promotes the survival of Staphylococcus aureus in coculture with Pseudomonas aeruginosa, an effect that is independent of its metal-sequestering function. In this work, we sought to understand how CP modulates this interspecies interaction by evaluating the transcriptional responses of P. aeruginosa and S. aureus to CP and metal limitation in cocultures. Our study revealed that CP attenuates the ability of P. aeruginosa to attack S. aureus with anti-staphylococcal factors and enhances the capacity of S. aureus to withstand this assault, effects that are not recapitulated by metal limitation. This work provides new understanding of how CP modulates microbial interactions that are relevant to human health.

microbiology↗

bioRxiv2024

The Pseudomonas aeruginosa PrrF sRNAs promote biofilm formation at body temperature

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes both acute and chronic infections in vulnerable populations. Treatment of P. aeruginosa infections is increasingly challenging due to multi-drug resistance as well as biofilm formation that further increases antibiotic tolerance. Iron, which is sequestered by the host innate immune system, is also a key nutrient that required for P. aeruginosa biofilm formation. Previous work showed that the iron-responsive PrrF small regulatory RNAs (sRNAs), which are key to P. aeruginosas iron starvation response and required for virulence in murine lung infection, are dispensable for biofilm formation. However, these studies were performed using flow-cell biofilms at room temperature. Here we demonstrate a temperature dependency for PrrF in P. aeruginosa biofilm formation - the genes for these sRNAs are required for optimal biofilm formation at 37{degrees}C but not 25{degrees}C. We further show that the {Delta}prrF mutant lacks a yet-to-be identified surface appendage that is produced at 37{degrees}C but not 25{degrees}C. These studies demonstrate that the importance of the PrrF sRNAs in P. aeruginosa biofilm formation at body temperature and reveal a previously under-appreciated role of temperature in iron homeostasis and P. aeruginosa biofilm physiology. IMPORTANCEBiofilm formation is a critical aspect of pathogenesis for many microbial pathogens as it confers increased tolerance to the host immune system and antimicrobial treatments. Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms during infection, resulting in antimicrobial tolerance and treatment failure. Iron is a known requirement for P. aeruginosa biofilm formation, yet the precise role of iron homeostasis in biofilm physiology remains unclear. Here we show that temperature alters the requirement for the PrrF small regulatory RNAs, key components of P. aeruginosas iron starvation response, for biofilm formation. Specifically, PrrF is required for the optimal formation of biofilms in flow cells at 37{degrees}C but not 25{degrees}C, yet most flow-cell biofilm studies are conducted at 25{degrees}C. These results demonstrate a previously under-appreciated role of temperature in P. aeruginosa biofilm physiology.

microbiology↗

bioRxiv2023

The heme-responsive PrrH sRNA regulates Pseudomonas aeruginosa pyochelin gene expression

Pseudomonas aeruginosa is an opportunistic pathogen that requires iron for growth and virulence, yet this nutrient is sequestered by the innate immune system during infection. When iron is limiting, P. aeruginosa expresses the PrrF1 and PrrF2 small regulatory RNAs (sRNAs), which post-transcriptionally repress expression of non-essential iron-containing proteins thus sparing this nutrient for more critical processes. The genes for the PrrF1 and PrrF2 sRNAs are arranged in tandem on the chromosome, allowing for the transcription of a longer heme-responsive sRNA, termed PrrH. While the functions of PrrF1 and PrrF2 have been studied extensively, the role of PrrH in P. aeruginosa physiology and virulence is not well understood. In this study, we performed transcriptomic and proteomic studies to identify the PrrH regulon. In shaking cultures, the pyochelin synthesis proteins were increased in two distinct prrH mutants compared to wild type, while the mRNAs for these proteins were not affected by prrH mutation. We identified complementarity between the PrrH sRNA and sequence upstream of the pchE mRNA, suggesting potential for PrrH to directly regulate expression of genes for pyochelin synthesis. We further showed that pchE mRNA levels were increased in the prrH mutants when grown in static but not shaking conditions. Moreover, we discovered controlling for the presence of light was critical for examining the impact of PrrH on pchE expression. As such, our study reports on the first likely target of the PrrH sRNA and highlights key environmental variables that will allow for future characterization of PrrH function. ImportanceIn the human host, iron is predominantly in the form of heme, which Pseudomonas aeruginosa can acquire as an iron source during infection. We previously showed that the iron-responsive PrrF sRNAs are critical for mediating iron homeostasis during P. aeruginosa infection; however the function of the heme-responsive PrrH sRNA remains unclear. In this study, we identified genes for pyochelin siderophore biosynthesis, which mediate uptake of inorganic iron, as a novel target of PrrH regulation. This study therefore highlights a novel relationship between heme availability and siderophore biosynthesis in P. aeruginosa.

microbiology↗

bioRxiv2022

Pseudomonas aeruginosa mediates PqsA-dependent iron regulation of the RsmY and RsmZ sRNAs in static conditions

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes acute and chronic lung infection in compromised hosts. During infection, the host innate immune system restricts iron to limit microbial growth. In response, P. aeruginosa induces expression of numerous virulence genes. Recently, our lab showed that some virulence factors are responsive to iron limitation in static but not shaking growth conditions, the former of which is likely to mimic growth in the chronically-infected lung. One of these novel iron-responsive factors is the HSI-2-type six secretion system (T6SS), which is also induced during chronic infection. Iron regulation of T6SS was partially impacted by deletion of the iron-responsive PrrF sRNA and completely dependent upon the Pseudomonas quinolone signal (PQS) biosynthetic gene pqsA. Here, we analyzed the impact of iron on the expression of two small regulatory RNAs (sRNAs), RsmY and RsmZ, that activate expression of T6SS by sequestering the RsmA translation inhibitor. Our results demonstrate that iron starvation induces expression of RsmY and RsmZ in static but not shaking cultures. We further show that this induction occurs through the rsmY and rsmZ promoters and is dependent upon PqsA. We identified interrupted palindromes in the rsmY and rsmZ promoters as putative PqsR binding sites, and disruption of these sites eliminated iron-dependent regulation of rsmY and rsmZ promoter activity. To determine if iron-dependent regulation of the Rsm sRNAs is likely responsible for iron regulation of HSI-2 T6SS, we constructed translational and transcriptional reporters of the hsiA2 T6SS gene. Analysis of these reporters revealed robust PqsA-mediated iron regulation of the transcriptional reporter, as well as modest PrrF-dependent iron regulation of the translational reporter. Taken together, our results show novel iron regulatory pathways that are promoted by static growth, highlighting the importance of studying regulatory mechanisms in static communities that are likely more representative of chronic P. aeruginosa infections. IMPORTANCEIron is a central component of various bacterial metabolic pathways making it an important host acquired nutrient for pathogens to establish infection. Previous iron regulatory studies primaried relied on shaking bacterial cultures; while these ensure cultural homogeneity they do not reflect growth conditions during infection. We recently showed that static growth of Pseudomonas aeruginosa promotes iron-dependent regulation of a type six secretion system (T6SS), a virulence factor that is induced during chronic infections. In the current study, we found that static growth also promotes iron-dependent regulation of the RsmY and RsmZ sRNAs, which are global regulators that affect T6SS during chronic P. aeruginosa lung infection. Hence, our work demonstrates the Rsm sRNAs as potential effectors of iron regulation during static growth that may also be relevant in chronic infection.

microbiology↗

bioRxiv2021

The human innate immune protein calprotectin elicits a multi-metal starvation response in Pseudomonas aeruginosa

To combat infections, the mammalian host limits availability of essential transition metals such as iron (Fe), zinc (Zn), and manganese (Mn) in a strategy termed "nutritional immunity". The innate immune protein calprotectin (CP) contributes to nutritional immunity by sequestering these metals to exert antimicrobial activity against a broad range of microbial pathogens. One such pathogen is Pseudomonas aeruginosa, which causes opportunistic infections in vulnerable populations including individuals with cystic fibrosis. CP was previously shown to withhold Fe(II) and Zn(II) from P. aeruginosa and induce Fe- and Zn-starvation responses in this pathogen. In this work, we performed quantitative, label-free proteomics to further elucidate how CP impacts metal homeostasis pathways in P. aeruginosa. We report that CP induces an incomplete Fe-starvation response, as many Fe-containing proteins that are repressed by Fe limitation are not affected by CP treatment. The Zn-starvation response elicited by CP seems to be more complete than the Fe-starvation response and includes increases in Zn transporters and Zn-independent proteins. CP also induces the expression of membrane-modifying proteins, and metal-depletion studies indicate this response results from the sequestration of multiple metals. Moreover, the increased expression of membrane-modifying enzymes upon CP treatment correlates with increased resistance to polymyxin B. Thus, response of P. aeruginosa to CP treatment includes both single and multi-metal starvation responses and includes many factors related to virulence potential, broadening our understanding of this pathogens interaction with the host. ImportanceTransition metals are critical for growth and infection by all pathogens, and the innate immune system withholds these metals from pathogens to limit their growth in a strategy termed "nutritional immunity". While multi-metal depletion by the host is appreciated, the majority of metal depletion studies have focused on individual metald. Here we use the innate immune protein calprotectin (CP), which complexes with several metals including iron (Fe), zinc (Zn), and manganese (Mn), and the opportunistic pathogen Pseudomonas aeruginosa to investigate multi-metal starvation. Using an unbiased label-free proteomics response, we demonstrate that multi-metal withholding by CP induces a regulatory response that is not merely additive of individual metal starvation responses, including the induction of Lipid A modification enzymes.

microbiology↗