David L. Erickson

Pseudomonas aeruginosa Quorum-Sensing Systems May Control Virulence Factor Expression in the Lungs of Patients with Cystic Fibrosis

ABSTRACT Individuals with cystic fibrosis (CF) are commonly colonized with Pseudomonas aeruginosa. The chronic infections caused by P. aeruginosa are punctuated by acute exacerbations of the lung disease, which lead to significant morbidity and mortality. As regulators of virulence determinants, P. aeruginosa quorum-sensing systems may be active in the chronic lung infections associated with CF. We have examined the levels of autoinducer molecules and transcript accumulation from the bacterial populations found in the lungs of patients with CF. We detected biologically active levels of N-(3-oxododecanoyl)-l-homoserine (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL) in sputum from CF patients. Interestingly, it appears that C4-HSL is less frequently detected than 3-oxo-C12-HSL in the lungs of patients with CF. We also examined the transcription of the autoinducer synthase gene lasI and showed that it is frequently expressed in the lungs of patients with CF. We observed a significant correlation between the expression of lasI and four target genes of the Las quorum-sensing system. Taken together, our results indicate that quorum-sensing systems are active and may control virulence factor expression in the lungs of patients with CF.

Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster.

ABSTRACT The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.

The Stringent Response Is Essential for Pseudomonas aeruginosa Virulence in the Rat Lung Agar Bead and Drosophila melanogaster Feeding Models of Infection

ABSTRACT The stringent response is a regulatory system that allows bacteria to sense and adapt to nutrient-poor environments. The central mediator of the stringent response is the molecule guanosine 3′,5′-bispyrophosphate (ppGpp), which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT. Our laboratory previously demonstrated that a relA mutant of Pseudomonas aeruginosa, the principal cause of lung infections in cystic fibrosis patients, was attenuated in virulence in a Drosophila melanogaster feeding model of infection. In this study, we examined the role of spoT in P. aeruginosa virulence. We generated an insertion mutation in spoT within the previously constructed relA mutant, thereby producing a ppGpp-devoid strain. The relA spoT double mutant was unable to establish a chronic infection in D. melanogaster and was also avirulent in the rat lung agar bead model of infection, a model in which the relA mutant is fully virulent. Synthesis of the virulence determinants pyocyanin, elastase, protease, and siderophores was impaired in the relA spoT double mutant. This mutant was also defective in swarming and twitching, but not in swimming motility. The relA spoT mutant and, to a lesser extent, the relA mutant were less able to withstand stresses such as heat shock and oxidative stress than the wild-type strain PAO1, which may partially account for the inability of the relA spoT mutant to successfully colonize the rat lung. Our results indicate that the stringent response, and SpoT in particular, is a crucial regulator of virulence processes in P. aeruginosa.

Bacterial communities associated with flea vectors of plague.

ABSTRACT The microbial flora associated with fleas may affect their ability to transmit specific pathogens, including Yersinia pestis, and also could be used to develop paratransgenesis-based approaches to interfere with transmission. To begin addressing this hypothesis, the microbial flora associated with the relatively efficient Y. pestis vectors Xenopsylla cheopis (Rothschild) (Siphonaptera: Pulicidae) and Oropsylla montana (Baker) (Siphonaptera: Ceratophyllidae), and the inefficient vector Ctenocephalides felis felis (Bouché) (Siphonaptera: Pulicidae) were investigated using polymerase chain reaction amplification of 16S rDNA genes. DNA sequencing revealed that these species harbor distinct communities of microbial flora and suggest that Acinetobacter sp. might be used in developing anti-transmission strategies.

PhoP and OxyR transcriptional regulators contribute to Yersinia pestis virulence and survival within Galleria mellonella.

The virulence of Yersinia pestis KIM6+ was compared with multiple isolates of Yersinia pseudotuberculosis and Yersinia enterocolitica toward larvae of the greater wax moth Galleria mellonella. Although Y.xa0pestis and Y.xa0pseudotuberculosis were able to cause lethal infection in G.xa0mellonella, these species appeared less virulent than the majority of Y.xa0enterocolitica strains tested. Y.xa0pestis survived primarily within hemocytes of G.xa0mellonella, and induced a strong antibacterial peptide response that lasted for at least 3 days in surviving larvae. Immunization with dead bacteria to induce an antibacterial response led to increased survival of the larvae following infection. Mutant strains lacking the either phoP or oxyR, which were less resistant to antibacterial peptides and hydrogen peroxide respectively, were attenuated and restoration of the wild-type genes on plasmids restored virulence. Our results indicate that the Y.xa0pseudotuberculosis-Y. pestis lineage is not as virulent toward G.xa0mellonella as are the majority of Y.xa0enterocolitica isolates. Further, we have shown that G.xa0mellonella is a useful infection model for analyzing Y.xa0pestis host-pathogen interactions, and antibacterial peptide resistance mediated by phoP and reactive oxygen defense mediated by oxyR are important for Y.xa0pestis infection of this insect.

Gene Expression Analysis of Xenopsylla cheopis (Siphonaptera: Pulicidae) Suggests a Role for Reactive Oxygen Species in Response to Yersinia pestis Infection

ABSTRACT n Fleas are vectors for a number of pathogens including Yersinia pestis, yet factors that govern interactions between fleas and Y. pestis are not well understood. Examining gene expression changes in infected fleas could reveal pathways that affect Y. pestis survival in fleas and subsequent transmission. We used suppression subtractive hybridization to identify genes that are induced in Xenopsylla cheopis (Rothschild) (Siphonaptera: Pulicidae) in response to oral or hemocoel infection with Y. pestis. Overall, the transcriptional changes we detected were very limited. We identified several genes that are likely involved in the production or removal of reactive oxygen species (ROS). Midgut ROS levels were higher in infected fleas and antioxidant treatment before infection reduced ROS levels and resulted in higher bacterial loads. An ROS-sensitive mutant strain of Y. pestis lacking the OxyR transcriptional regulator showed reduced growth early after infection. Our results indicate that ROS may limit Y. pestis early colonization of fleas and that bacterial strategies to overcome ROS may enhance transmission.

Lipopolysaccharide Biosynthesis Genes of Yersinia pseudotuberculosis Promote Resistance to Antimicrobial Chemokines

Antimicrobial chemokines (AMCs) are a recently described family of host defense peptides that play an important role in protecting a wide variety of organisms from bacterial infection. Very little is known about the bacterial targets of AMCs or factors that influence bacterial susceptibility to AMCs. In an effort to understand how bacterial pathogens resist killing by AMCs, we screened Yersinia pseudotuberculosis transposon mutants for those with increased binding to the AMCs CCL28 and CCL25. Mutants exhibiting increased binding to AMCs were subjected to AMC killing assays, which revealed their increased sensitivity to chemokine-mediated cell death. The majority of the mutants exhibiting increased binding to AMCs contained transposon insertions in genes related to lipopolysaccharide biosynthesis. A particularly strong effect on susceptibility to AMC mediated killing was observed by disruption of the hldD/waaF/waaC operon, necessary for ADP-L-glycero-D-manno-heptose synthesis and a complete lipopolysaccharide core oligosaccharide. Periodate oxidation of surface carbohydrates also enhanced AMC binding, whereas enzymatic removal of surface proteins significantly reduced binding. These results suggest that the structure of Y. pseudotuberculosis LPS greatly affects the antimicrobial activity of AMCs by shielding a protein ligand on the bacterial cell surface.

Bovine CCL28 Mediates Chemotaxis via CCR10 and Demonstrates Direct Antimicrobial Activity against Mastitis Causing Bacteria

In addition to the well characterized function of chemokines in mediating the homing and accumulation of leukocytes to tissues, some chemokines also exhibit potent antimicrobial activity. Little is known of the potential role of chemokines in bovine mammary gland health and disease. The chemokine CCL28 has previously been shown to play a key role in the homing and accumulation of IgA antibody secreting cells to the lactating murine mammary gland. CCL28 has also been shown to act as an antimicrobial peptide with activity demonstrated against a wide range of pathogens including bacteria, fungi and protozoans. Here we describe the cloning and function of bovine CCL28 and document the concentration of this chemokine in bovine milk. Bovine CCL28 was shown to mediate cellular chemotaxis via the CCR10 chemokine receptor and exhibited antimicrobial activity against a variety of bovine mastitis causing organisms. The concentration of bovine CCL28 in milk was found to be highly correlated with the lactation cycle. Highest concentrations of CCL28 were observed soon after parturition, with levels decreasing over time. These results suggest a potential role for CCL28 in the prevention/resolution of bovine mastitis.

Genome-wide identification of fitness factors in mastitis-associated Escherichia coli

ABSTRACT Virulence factors of mammary pathogenic Escherichia coli (MPEC) have not been identified, and it is not known how bacterial gene content influences the severity of mastitis. Here, we report a genome-wide identification of genes that contribute to fitness of MPEC under conditions relevant to the natural history of the disease. A highly virulent clinical isolate (M12) was identified that killed Galleria mellonella at low infectious doses and that replicated to high numbers in mouse mammary glands and spread to spleens. Genome sequencing was combined with transposon insertion site sequencing to identify MPEC genes that contribute to growth in unpasteurized whole milk, as well as during G. mellonella and mouse mastitis infections. These analyses show that strain M12 possesses a unique genomic island encoding a group III polysaccharide capsule that greatly enhances virulence in G. mellonella. Several genes appear critical for MPEC survival in both G. mellonella and in mice, including those for nutrient-scavenging systems and resistance to cellular stress. Insertions in the ferric dicitrate receptor gene fecA caused significant fitness defects under all conditions (in milk, G. mellonella, and mice). This gene was highly expressed during growth in milk. Targeted deletion of fecA from strain M12 caused attenuation in G. mellonella larvae and reduced growth in unpasteurized cows milk and lactating mouse mammary glands. Our results confirm that iron scavenging by the ferric dicitrate receptor, which is strongly associated with MPEC strains, is required for MPEC growth and may influence disease severity in mastitis infections. IMPORTANCE Mastitis caused by E. coli inflicts substantial burdens on the health and productivity of dairy animals. Strains causing mastitis may express genes that distinguish them from other E. coli strains and promote infection of mammary glands, but these have not been identified. Using a highly virulent strain, we employed genome-wide mutagenesis and sequencing to discover genes that contribute to mastitis. This extensive data set represents a screen for mastitis-associated E. coli fitness factors and provides the following contributions to the field: (i) global comparison of genes required for different aspects of mastitis infection, (ii) discovery of a unique capsule that contributes to virulence, and (iii) conclusive evidence for the crucial role of iron-scavenging systems in mastitis, particularly the ferric dicitrate transport system. Similar approaches applied to other mastitis-associated strains will uncover conserved targets for prevention or treatment and provide a better understanding of their relationship to other E. coli pathogens.

Yersinia pseudotuberculosis BarA-UvrY Two-Component Regulatory System Represses Biofilms via CsrB

The formation of biofilms by Yersinia pseudotuberculosis (Yptb) and Y. pestis requires the hmsHFRS genes, which direct production of a polysaccharide extracellular matrix (Hms-ECM). Despite possessing identical hmsHFRS sequences, Yptb produces much less Hms-ECM than Y. pestis. The regulatory influences that control Yptb Hms-ECM production and biofilm formation are not fully understood. In this study, negative regulators of biofilm production in Yptb were identified. Inactivation of the BarA/UvrY two-component system or the CsrB regulatory RNA increased binding of Congo Red dye, which correlates with extracellular polysaccharide production. These mutants also produced biofilms that were substantially more cohesive than the wild type strain. Disruption of uvrY was not sufficient for Yptb to cause proventricular blockage during infection of Xenopsylla cheopis fleas. However, this strain was less acutely toxic toward fleas than wild type Yptb. Flow cytometry measurements of lectin binding indicated that Yptb BarA/UvrY/CsrB mutants may produce higher levels of other carbohydrates in addition to poly-GlcNAc Hms-ECM. In an effort to characterize the relevant downstream targets of the BarA/UvrY system, we conducted a proteomic analysis to identify proteins with lower abundance in the csrB::Tn5 mutant strain. Urease subunit proteins were less abundant and urease enzymatic activity was lower, which likely reduced toxicity toward fleas. Loss of CsrB impacted expression of several potential regulatory proteins that may influence biofilms, including the RcsB regulator. Overexpression of CsrB did not alter the Congo-red binding phenotype of an rcsB::Tn5 mutant, suggesting that the effect of CsrB on biofilms may require RcsB. These results underscore the regulatory and compositional differences between Yptb and Y. pestis biofilms. By activating CsrB expression, the Yptb BarA/UvrY two-component system has pleiotropic effects that impact biofilm production and stability.