A rise in the frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021.

Introduction: Pseudomonas aeruginosa causes vision threatening keratitis. The LasR transcription factor regulates virulence factors in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study demonstrated 22% (n=101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive lasR mutants, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene. Methods: Keratitis isolates (n=399) were classified by sheen phenotype. The lasR gene was cloned from a subset of isolates, sequenced, and tested for loss of function or dominant-negative status based on an azocasein protease assay. A retrospective chart review compared outcomes of keratitis patients infected by sheen positive and negative isolates. Results: A significant increase in sheen positive isolates was observed between 1993 and 2021. Extracellular protease activity was reduced among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Retrospective analysis of patient information suggested significantly better visual outcomes for patients infected by sheen positive isolates. Discussion: These results indicate an increase in lasR mutations among keratitis isolates in the United States and suggest that endemic lasR mutants can cause keratitis.

Rise in frequency of lasR mutant Pseudomonas aeruginosa among keratitis isolates between 1993 and 2021.

Pseudomonas aeruginosa causes severe vision threatening keratitis. LasR is a transcription factor that regulates virulence associated genes in response to the quorum sensing molecule N-3-oxo-dodecanoyl-L-homoserine lactone. P. aeruginosa isolates with lasR mutations are characterized by an iridescent high sheen phenotype caused by a build-up of 2-heptyl-4-quinolone. A previous study indicated a high proportion (22 out of 101) of P. aeruginosa keratitis isolates from India between 2010 and 2016 were sheen positive and had mutations in the lasR gene, and the sheen phenotype correlated with worse clinical outcomes for patients. In this study, a longitudinal collection of P. aeruginosa keratitis isolates from Eastern North America were screened for lasR mutations by the sheen phenotype and sequencing of the lasR gene. A significant increase in the frequency of isolates with the sheen positive phenotype was observed in isolates between 1993 and 2021. Extracellular protease activity was lower among the sheen positive isolates and a defined lasR mutant. Cloned lasR alleles from the sheen positive isolates were loss of function or dominant negative and differed in sequence from previously reported ocular lasR mutant alleles. Insertion elements were present in a subset of independent isolates and may represent an endemic source from some of the isolates. Retrospective analysis of patient information suggested significantly better visual outcomes for patients with infected by sheen positive isolates. Together, these results indicate an increasing trend towards lasR mutations among keratitis isolates at a tertiary eye care hospital in the United States.

Xylose-Inducible Promoter Tools for Pseudomonas Species and Their Use in Implicating a Role for the Type II Secretion System Protein XcpQ in the Inhibition of Corneal Epithelial Wound Closure.

Tunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and Pseudomonas fluorescens The Pxut promoter, derived from the P. fluorescensxut operon, was incorporated into a broad-host-range pBBR1-based plasmid and was compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. Green fluorescent protein (GFP) fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate the cloning of genes toxic to E. coli to generate plasmids. The Pxut promoter was activated at a lower inducer concentration than PBAD in P. fluorescens, and higher gfp levels were achieved using Pxut Flow cytometry analysis indicated that Pxut was leakier than PBAD in the Pseudomonas species tested but was expressed in a higher proportion of cells when induced. d-Xylose as a sole carbon source did not support the growth of P. aeruginosa or P. fluorescens and is less expensive than many other commonly used inducers, which could facilitate large-scale applications. The efficacy of this system was demonstrated by its use to reveal a role for the P. aeruginosa type II secretion system gene xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species.IMPORTANCEPseudomonas species are enormously important in human infections, in biotechnology, and as model systems for investigating basic science questions. In this study, we have developed a xylose-inducible promoter system, evaluated it in P. aeruginosa and P. fluorescens, and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type II secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

Use of Collagen Binding Domains to Deliver Molecules to the Cornea.

Purpose: The combined activity of the tear film and blinking is remarkably efficient at removal of foreign materials from the ocular surface. This has prevented the use of certain classes of drugs for the treatment of ocular surface problems. We propose that the use of peptide and protein domains that bind to moieties on the cornea could be used to deliver therapeutics by anchoring the drugs on the ocular surface long enough to provide therapeutic effects. Methods: In this study, we evaluated 4 different collagen binding domains fused to bacterial ß-galactosidase for delivery of a reporter protein to collagen I and collagen IV-coated plates, rabbit corneas, and Herpes simplex virus (HSV-1) infected mouse corneas. Results: All 4 domains bound to collagen I and IV in vitro, whereas only a 10 amino acid (AA) sequence from bovine von Willebrand factor (vWF) and a 215 AA collagen binding domain from the bacterial protein ColH efficiently bound to abraded rabbit corneas. To test binding to corneas in a clinically relevant model, we assessed binding of the vWF collagen binding peptide fusions to HSV-1 infected mouse corneas. We observed that the vWF derived peptide mediated attachment to infected corneas, whereas the reporter protein without a collagen binding domain did not bind. Conclusions: Moving forward, the vWF collagen binding peptide could be used as an anchor to deliver therapeutics to prevent scarring and vision loss from damaged corneal surfaces due to disease and inflammation.

Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.

Thermoregulation of Prodigiosin Biosynthesis by Serratia marcescens is Controlled at the Transcriptional Level and Requires HexS.

Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level.Several biotypes of the Gram-negative bacterium Serratia marcescens produce the tri-pyrole pigment and secondary metabolite prodigiosin. The biological activities of this pigment have therapeutic potential. For over half a century it has been known that biosynthesis of prodi giosin is inhibited when bacteria are grown at elevated temperatures, yet the fundamental mechanism underlying this thermoregulation has not been characterized. In this study, chromosomal and plasmid-borne luxCDABE transcriptional reporters revealed reduced transcription of the prodigiosin biosynthetic operon at 37°C compared to 30°C indicating transcriptional control of pigment production. Moreover, induced expression of the prodigiosin biosynthetic operon at 37°C was able to produce pigmented colonies and cultures demonstrating that physiological conditions at 37°C allow prodigiosin production and indicating that post-transcriptional control is not a major contributor to the thermoregulation of prodigiosin pigmentation. Genetic experiments support the model that the HexS transcription factor is a key contributor to thermoregulation of pigmentation, whereas CRP plays a minor role, and a clear role for EepR and PigP was not observed. Together, these data indicate that thermoregulation of prodigiosin production at elevated temperatures is controlled largely, if not exclusively, at the transcriptional level.

Serralysin family metalloproteases protects Serratia marcescens from predation by the predatory bacteria Micavibrio aeruginosavorus.

Micavibrio aeruginosavorus is an obligate Gram-negative predatory bacterial species that feeds on other Gram-negative bacteria by attaching to the surface of its prey and feeding on the prey's cellular contents. In this study, Serratia marcescens with defined mutations in genes for extracellular cell structural components and secreted factors were used in predation experiments to identify structures that influence predation. No change was measured in the ability of the predator to prey on S. marcescens flagella, fimbria, surface layer, prodigiosin and phospholipase-A mutants. However, higher predation was measured on S. marcescens metalloprotease mutants. Complementation of the metalloprotease gene, prtS, into the protease mutant, as well as exogenous addition of purified serralysin metalloprotease, restored predation to wild type levels. Addition of purified serralysin also reduced the ability of M. aeruginosavorus to prey on Escherichia coli. Incubating M. aeruginosavorus with purified metalloprotease was found to not impact predator viability; however, pre-incubating prey, but not the predator, with purified metalloprotease was able to block predation. Finally, using flow cytometry and fluorescent microscopy, we were able to confirm that the ability of the predator to bind to the metalloprotease mutant was higher than that of the metalloprotease producing wild-type. The work presented in this study shows that metalloproteases from S. marcescens could offer elevated protection from predation.

An IgaA/UmoB Family Protein from Serratia marcescens Regulates Motility, Capsular Polysaccharide Biosynthesis, and Secondary Metabolite Production.

Secondary metabolites are an important source of pharmaceuticals and key modulators of microbe-microbe interactions. The bacterium Serratia marcescens is part of the Enterobacteriaceae family of eubacteria and produces a number of biologically active secondary metabolites. In this study, we screened for novel regulators of secondary metabolites synthesized by a clinical isolate of S. marcescens and found mutations in a gene for an uncharacterized UmoB/IgaA family member here named gumB Mutation of gumB conferred a severe loss of the secondary metabolites prodigiosin and serratamolide. The gumB mutation conferred pleiotropic phenotypes, including altered biofilm formation, highly increased capsular polysaccharide production, and loss of swimming and swarming motility. These phenotypes corresponded to transcriptional changes in fimA, wecA, and flhD Unlike other UmoB/IgaA family members, gumB was found to be not essential for growth in S. marcescens, yet igaA from Salmonella enterica, yrfF from Escherichia coli, and an uncharacterized predicted ortholog from Klebsiella pneumoniae complemented the gumB mutant secondary metabolite defects, suggesting highly conserved function. These data support the idea that UmoB/IgaA family proteins are functionally conserved and extend the known regulatory influence of UmoB/IgaA family proteins to the control of competition-associated secondary metabolites and biofilm formation.IMPORTANCE IgaA/UmoB family proteins are found in members of the Enterobacteriaceae family of bacteria, which are of environmental and public health importance. IgaA/UmoB family proteins are thought to be inner membrane proteins that report extracellular stresses to intracellular signaling pathways that respond to environmental challenge. This study introduces a new member of the IgaA/UmoB family and demonstrates a high degree of functional similarity between IgaA/UmoB family proteins. Moreover, this study extends the phenomena controlled by IgaA/UmoB family proteins to include the biosynthesis of antimicrobial secondary metabolites.

Widespread Fosfomycin Resistance in Gram-Negative Bacteria Attributable to the Chromosomal fosA Gene.

Fosfomycin is a decades-old antibiotic which is being revisited because of its perceived activity against many extensively drug-resistant Gram-negative pathogens. FosA proteins are Mn2+ and K+-dependent glutathione S-transferases which confer fosfomycin resistance in Gram-negative bacteria by conjugation of glutathione to the antibiotic. Plasmid-borne fosA variants have been reported in fosfomycin-resistant Escherichia coli strains. However, the prevalence and distribution of fosA in other Gram-negative bacteria are not known. We systematically surveyed the presence of fosA in Gram-negative bacteria in over 18,000 published genomes from 18 Gram-negative species and investigated their contribution to fosfomycin resistance. We show that FosA homologues are present in the majority of genomes in some species (e.g., Klebsiella spp., Enterobacter spp., Serratia marcescens, and Pseudomonas aeruginosa), whereas they are largely absent in others (e.g., E. coli, Acinetobacter baumannii, and Burkholderia cepacia). FosA proteins in different bacterial pathogens are highly divergent, but key amino acid residues in the active site are conserved. Chromosomal fosA genes conferred high-level fosfomycin resistance when expressed in E. coli, and deletion of chromosomal fosA in S. marcescens eliminated fosfomycin resistance. Our results indicate that FosA is encoded by clinically relevant Gram-negative species and contributes to intrinsic fosfomycin resistance.IMPORTANCE There is a critical need to identify alternate approaches to treat infections caused by extensively drug-resistant (XDR) Gram-negative bacteria. Fosfomycin is an old antibiotic which is routinely used for the treatment of urinary tract infections, although there is substantial interest in expanding its use to systemic infections caused by XDR Gram-negative bacteria. In this study, we show that fosA genes, which encode dimeric Mn2+- and K+-dependent glutathione S-transferase, are widely distributed in the genomes of Gram-negative bacteria-particularly those belonging to the family Enterobacteriaceae-and confer fosfomycin resistance. This finding suggests that chromosomally located fosA genes represent a vast reservoir of fosfomycin resistance determinants that may be transferred to E. coli Furthermore, they suggest that inhibition of FosA activity may provide a viable strategy to potentiate the activity of fosfomycin against XDR Gram-negative bacteria.

SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens.

Serralysin-like proteases are found in a wide variety of bacteria. These metalloproteases are frequently implicated in virulence and are members of the widely conserved RTX-toxin family. We identified a serralysin-like protease in the genome of a clinical isolate of Serratia marcescens that is highly similar to the canonical serralysin protein, PrtS. This gene was named serralysin-like protease E, SlpE, and was found in the majority (67%) of tested clinical isolates, but was absent from most tested non-clinical isolates including the insect pathogen and reference S. marcescens strain Db11. Purified recombinant SlpE exhibited calcium-dependent protease activity similar to metalloproteases PrtS and SlpB. Induction of slpE in the low-protease-producing S. marcescens strain PIC3611 highly elevated extracellular protease activity, and extracellular secretion required the lipD type 1 secretion system gene. Transcription of slpE was highly reduced in an eepR transcription factor mutant. Mutation of the slpE gene in a highly proteolytic clinical isolate reduced its protease activity, and evidence suggests that SlpE confers cytotoxicity of S. marcescens to the A549 airway carcinoma cell line. Together, these data reveal SlpE to be an EepR-regulated cytotoxic metalloprotease associated with clinical isolates of an important opportunistic pathogen.

Suppressor analysis of eepR mutant defects reveals coordinate regulation of secondary metabolites and serralysin biosynthesis by EepR and HexS.

The EepR transcription factor positively regulates secondary metabolites and tissue-damaging metalloproteases. To gain insight into mechanisms by which EepR regulates pigment and co-regulated factors, genetic suppressor analysis was performed. Suppressor mutations that restored pigment to the non-pigmented ∆eepR mutant mapped to the hexS ORF. Mutation of hexS also restored haemolysis, swarming motility and protease production to the eepR mutant. HexS is a known direct and negative regulator of secondary metabolites in Serratia marcescens and is a LysR family regulator and an orthologue of LrhA. Here, we demonstrate that HexS directly controls eepR and the serralysin gene prtS. EepR was shown to directly regulate eepR expression but indirectly regulate hexS expression. Together, these data indicate that EepR and HexS oppose each other in controlling stationary phase-associated molecules and enzymes.