Antibody-mediated immunological memory correlates with long-term Lyme veterinary vaccine protection in mice.

Lyme disease, caused by the bacterium Borrelia burgdorferi, is the most common tick-borne illness in the United States. Despite the rise in Lyme disease incidence, there is no vaccine against B. burgdorferi approved for human use. Little is known about the immune correlates of protection needed to prevent Lyme disease. In this work, a mouse model was used to characterize the immune response and compare the protection provided by two USDA-approved vaccines for use in canines: Duramune (bacterin vaccine) and Vanguard crLyme (subunit vaccine composed of two outer surface proteins, OspA and OspC). C3H/HeNCrl mice were immunized with two doses of either Duramune or Vanguard, and immune responses and protection against B. burgdorferi were assessed in short (35 days) and long-term (120 days) studies. Flow cytometry, ELISPOT detection of antibody-producing cells, and antibody affinity studies were performed to identify correlates of vaccine-mediated protection. Both vaccines induced humoral responses, with high IgG titers against B. burgdorferi. However, the levels of anti-B. burgdorferi antibodies decayed over time in Vanguard-vaccinated mice. While both vaccines triggered the production of antibodies against both OspA and OspC, antibody levels against these proteins were also lower in Vanguard-vaccinated mice 120 days post-vaccination. Both vaccines only provided partial protection against B. burgdorferi at the dose used in this model. The protection provided by Duramune was superior to Vanguard 120 days post-vaccination, and was characterized by higher antibody titers, higher abundance of long-lived plasma cells, and higher avidity antibodies than Vanguard. Overall, these studies provide insights into the importance of the humoral memory response to veterinary vaccines against Lyme disease and will help inform the development of future human vaccines.

Frontline Science: Myeloid cell-specific deletion of Cebpb decreases sepsis-induced immunosuppression in mice.

Sepsis inflammation accelerates myeloid cell generation to compensate for rapid mobilization of the myeloid progenitors from bone marrow. This inflammation-driven myelopoiesis, however, generates myeloid progenitors with immunosuppressive functions that are unable to differentiate into mature, innate immune cells. The myeloid-derived suppressor cells (MDSCs) expand markedly in the later phases of sepsis, suppress both innate and adaptive immunity, and thus, elevate mortality. Using a murine model with myeloid-restricted deletion of the C/EBPß transcription factor, we show that sepsis-induced generation of MDSCs depends on C/EBPß. C/EBPß myeloid cell-deficient mice did not generate MDSCs or develop immunosuppression and survived sepsis. However, septic mice still generated Gr1+CD11b+ myeloid progenitors at the steady-state levels similar to the control sham mice, suggesting that C/EBPß is not involved in healthy, steady-state myelopoiesis. C/EBPß-deficient Gr1+CD11b+ cells generated fewer monocyte- and granulocyte-like colonies than control mice did, indicating reduced proliferation potential, but differentiated normally in response to growth factors. Adoptive transfer of C/EBPß-deficient Gr1+CD11b+ cells from late septic mice exacerbated inflammation in control mice undergoing early sepsis, confirming they were not immunosuppressive. These results show that C/EBPß directs a switch from proinflammatory to repressor myeloid cells and identifies a novel treatment target.

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner.

Pseudomonas aeruginosa is an important pathogen of the immunocompromised, causing both acute and chronic infections. In cystic fibrosis (CF) patients, P. aeruginosa causes chronic disease. The impressive sensory network of P. aeruginosa allows the bacterium to sense and respond to a variety of stimuli found in diverse environments. Transcriptional regulators, including alternative sigma factors and response regulators, integrate signals changing gene expression, allowing P. aeruginosa to cause infection. The two-component transcriptional regulator AlgR is important in P. aeruginosa pathogenesis in both acute and chronic infections. In chronic infections, AlgR and the alternative sigma factor AlgU activate the genes responsible for alginate production. Previous work demonstrated that AlgU controls rsmA expression. RsmA is a posttranscriptional regulator that is antagonized by two small RNAs, RsmY and RsmZ. In this work, we demonstrate that AlgR directly activates rsmA expression from the same promoter as AlgU. In addition, phosphorylation was not necessary for AlgR activation of rsmA using algR and algZ mutant strains. AlgU and AlgR appear to affect the antagonizing small RNAs rsmY and rsmZ indirectly. RsmA was active in a mucA22 mutant strain using leader fusions of two RsmA targets, tssA1 and hcnA AlgU and AlgR were necessary for posttranscriptional regulation of tssA1 and hcnA Altogether, our work demonstrates that the alginate regulators AlgU and AlgR are important in the control of the RsmA posttranscriptional regulatory system. These findings suggest that RsmA plays an unknown role in mucoid strains due to AlgU and AlgR activities.IMPORTANCE P. aeruginosa infections are difficult to treat and frequently cause significant mortality in CF patients. Understanding the mechanisms of persistence is important. Our work has demonstrated that the alginate regulatory system also significantly impacts the posttranscriptional regulator system RsmA/Y/Z. We demonstrate that AlgR directly activates rsmA expression, and this impacts the RsmA regulon. This leads to the possibility that the RsmA/Y/Z system plays a role in helping P. aeruginosa persist during chronic infection. In addition, this furthers our understanding of the reach of the alginate regulators AlgU and AlgR.

Pseudomonas aeruginosa AlgU Contributes to Posttranscriptional Activity by Increasing rsmA Expression in a mucA22 Strain.

UNLABELLED: Pseudomonas aeruginosa thrives in multiple environments and is capable of causing life-threatening infections in immunocompromised patients. RsmA is a posttranscriptional regulator that controls virulence factor production and biofilm formation. In this study, we investigated the expression and activity of rsmA and the protein that it encodes, RsmA, in P. aeruginosa mucA mutant strains, which are common in chronic infections. We determined that AlgU regulates a previously unknown rsmA promoter in P. aeruginosa Western blot analysis confirmed that AlgU controls rsmA expression in both a laboratory strain and a clinical isolate. RNase protection assays confirmed the presence of two rsmA transcripts and suggest that RpoS and AlgU regulate rsmA expression. Due to the increased amounts of RsmA in mucA mutant strains, a translational leader fusion of the RsmA target, tssA1, was constructed and tested in mucA, algU, retS, gacA, and rsmA mutant backgrounds to examine posttranscriptional activity. From these studies, we determined that RsmA is active in mucA22 mutants, suggesting a role for RsmA in mucA mutant strains. Taken together, we have demonstrated that AlgU controls rsmA transcription and is responsible for RsmA activity in mucA mutant strains. We propose that RsmA is active in P. aeruginosa mucA mutant strains and that RsmA also plays a role in chronic infections. IMPORTANCE: P. aeruginosa causes severe infections in immunocompromised patients. The posttranscriptional regulator RsmA is known to control virulence and biofilm formation. We identify a new rsmA promoter and determine that AlgU is important in the control of rsmA expression. Mutant mucA strains that are considered mucoid were used to confirm increased rsmA expression from the AlgU promoter. We demonstrate, for the first time, that there is RsmA activity in mucoid P. aeruginosa strains. Our work suggests that RsmA may play a role during chronic infections as well as acute infections.

Expression analysis of the Pseudomonas aeruginosa AlgZR two-component regulatory system.

Pseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory system were examined. Primer extension and S1 nuclease protection assays were used to identify two transcriptional initiation sites for algR within the algZ coding region, and two additional start sites were identified upstream of the algZ coding region. The two algR transcriptional start sites, RT1 and RT2, are directly regulated by AlgU, consistent with previous reports of increased algR expression in mucoid backgrounds, and RpoS additionally plays a role in algR transcription. The expression of the first algZ promoter, ZT1, is entirely dependent upon Vfr for expression, whereas Vfr, RpoS, or AlgU does not regulate the second algZ promoter, ZT2. Western blot, real-time quantitative PCR (RT-qPCR), and transcriptional fusion analyses show that algZR expression is Vfr dependent. The algZ and algR genes also are cotranscribed in both nonmucoid and mucoid backgrounds. Furthermore, algZR was found to be cotranscribed with hemCD by RT-PCR. RT-qPCR confirmed that hemC transcription in the PAO1 ΔalgZ mutant was 40% of the level of the wild-type strain. Taken together, these results indicate that algZR transcription involves multiple factors at multiple start sites that control individual gene expression as well as coexpression of this two-component system with heme biosynthetic genes.

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa.

BACKGROUND: Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ(22)) to initiate transcription of the alginate biosynthetic operon. RESULTS: In the current study, we mapped the mucE transcriptional start site, and determined that P(mucE) activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P(mucE) activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. CONCLUSIONS: Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.

Identification of the mutation responsible for the temperature-sensitive lipopolysaccharide O-antigen defect in the Pseudomonas aeruginosa cystic fibrosis isolate 2192.

Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the exopolysaccharide alginate and the loss of complete lipopolysaccharide (LPS). LPS is a major component of the Gram-negative outer membrane and is composed of lipid A, core oligosaccharide, and O antigen. In this report, we show that the LPS defect of the P. aeruginosa chronic infection isolate 2192 is temperature sensitive. When grown at 25°C, 2192 expresses serotype O1 LPS with a moderate chain length and in reduced amounts relative to those of a wild-type serotype O1 laboratory strain (stO1). In contrast, 2192 expresses no LPS O antigen when grown at 37°C. This is the first time that a temperature-sensitive defect in O-antigen production has been reported. Using complementation analyses with a constructed wbpM deletion mutant of stO1, we demonstrate that the temperature-sensitive O-antigen production defect in 2192 is due to a mutation in wbpM, which encodes a UDP-4,6-GlcNAc dehydratase involved in O-antigen synthesis. The mutation, a deletion of a single amino acid (V636) from the extreme C terminus of WbpM, renders the protein less stable than its wild-type counterpart. This residue of WbpM, which is critical for stability and function, is located outside of the recognized domains of the protein and may provide insight into the structure-function relationship of this enzyme, which is found in all 20 serotypes of P. aeruginosa. We also identify a promoter of wbpM, map a transcriptional start site of wbpM, and show that mucoidy plays a role in the loss of expression of high-molecular-weight LPS in this CF isolate.

Activation of the Pseudomonas aeruginosa AlgU regulon through mucA mutation inhibits cyclic AMP/Vfr signaling.

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute, invasive infections in immunocompromised individuals and chronic, persistent respiratory infections in individuals with cystic fibrosis (CF). The differential progression of acute or chronic infections involves the production of distinct sets of virulence factors. P. aeruginosa strains isolated from patients with acute respiratory infection are generally nonencapsulated and express a variety of invasive virulence factors, including flagella, the type III secretion system (T3SS), type IV pili (TFP), and multiple secreted toxins and degradative enzymes. Strains isolated from chronically infected CF patients, however, typically lack expression of invasive virulence factors and have a mucoid phenotype due to the production of an alginate capsule. The mucoid phenotype results from loss-of-function mutations in mucA, which encodes an anti-sigma factor that normally prevents alginate synthesis. Here, we report that the cyclic AMP/Vfr-dependent signaling (CVS) pathway is defective in mucA mutants and that the defect occurs at the level of vfr expression. The CVS pathway regulates the expression of multiple invasive virulence factors, including T3SS, exotoxin A, protease IV, and TFP. We further demonstrate that mucA-dependent CVS inhibition involves the alternative sigma factor AlgU (AlgT) and the response regulator AlgR but does not depend on alginate production. Our findings show that a single naturally occurring mutation leads to inverse regulation of virulence factors involved in acute and persistent infections. These results suggest that mucoid conversion and inhibition of invasive virulence determinants may both confer a selective advantage to mucA mutant strains of P. aeruginosa in the CF lung.

Pseudomonas aeruginosa AlgR controls cyanide production in an AlgZ-dependent manner.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections in individuals suffering from the genetic disorder cystic fibrosis. In P. aeruginosa, the transcriptional regulator AlgR controls a variety of virulence factors, including alginate production, twitching motility, biofilm formation, quorum sensing, and hydrogen cyanide (HCN) production. In this study, the regulation of HCN production was examined. Strains lacking AlgR or the putative AlgR sensor AlgZ produced significantly less HCN than did a nonmucoid isogenic parent. In contrast, algR and algZ mutants showed increased HCN production in an alginate-producing (mucoid) background. HCN production was optimal in a 5% O2 environment. In addition, cyanide production was elevated in bacteria grown on an agar surface compared to bacteria grown in planktonic culture. A conserved AlgR phosphorylation site (aspartate at amino acid position 54), which is required for surface-dependent twitching motility but not alginate production, was found to be critical for cyanide production. Nuclease protection mapping of the hcnA promoter identified a new transcriptional start site required for HCN production. A subset of clinical isolates that lack this start site produced small amounts of cyanide. Taken together, these data show that the P. aeruginosa hcnA promoter contains three transcriptional start sites and that HCN production is regulated by AlgZ and AlgR and is maximal under microaerobic conditions when the organism is surface attached.

Molecular and physiological effects of mycobacterial oxyR inactivation.

The majority of slow-growing mycobacteria have a functional oxyR, the central regulator of the bacterial oxidative stress response. In contrast, this gene has been inactivated during the evolution of Mycobacterium tuberculosis. Here we inactivated the oxyR gene in Mycobacterium marinum, an organism used to model M. tuberculosis pathogenesis. Inactivation of oxyR abrogated induction of ahpC, a gene encoding alkylhydroperoxide reductase, normally activated upon peroxide challenge. The absence of oxyR also resulted in increased sensitivity to the front-line antituberculosis drug isoniazid. Inactivation of oxyR in M. marinum did not affect either virulence in a fish infection model or survival in human macrophages. Our findings demonstrate, at the genetic and molecular levels, a direct role for OxyR in ahpC regulation in response to oxidative stress. Our study also indicates that oxyR is not critical for virulence in M. marinum. However, oxyR inactivation confers increased sensitivity to isonicotinic acid hydrazide, suggesting that the natural loss of oxyR in the tubercle bacillus contributes to the unusually high sensitivity of M. tuberculosis to isoniazid.

Identification of Mycobacterium marinum virulence genes using signature-tagged mutagenesis and the goldfish model of mycobacterial pathogenesis.

Mycobacterium marinum, a causative agent of fish tuberculosis, is one of the most closely related Mycobacterium species (outside the M. tuberculosis complex) to M. tuberculosis, the etiologic agent of human tuberculosis. Signature-tagged mutagenesis was used to identify genes of M. marinum required for in vivo survival in a goldfish model of mycobacterial pathogenesis. Screening the first 1008 M. marinum mutants led to the identification of 40 putative virulence mutants. DNA sequence analysis of these 40 mutants identified transposon insertions in 35 unique loci. Twenty-eight out of 33 (85%) loci encoding putative virulence genes have homologous genes in M. tuberculosis.