Pseudomonas aeruginosa is a Gram-negative bacterium implicated in several clinical contexts. In its association with immunocompromised hosts including cystic fibrosis patients, P. aeruginosa is able to exploit the host immune response to acquire key factors essential to its adaptation. As such, key virulence factors including the Type III Secretion System (T3SS), initially essential in acute infection, is reduced in its significance in chronic colonization. On the contrary, other phenotypes are essential for the altered priorities in chronic colonization. The signals of the host immune response initiating the phenotypic switch from the expression of acute virulence factors to chronic virulence factors have not been well defined. Additionally, the function of the type VI secretion system (T6SS), a protein secretion apparatus, in chronic infection has been well established. Clinical isolates obtained from acute and chronic P. aeruginosa infections suggested selective regulation of the T6SS, namely up regulation of the H3-T6SS in chronic infection. We used murine models of infection to understand the in vivo transcriptional regulation of the T6SS of PAO1. Itaconate, an anti-inflammatory metabolite generated by the host, selectively upregulated transcription of a H3-T6SS-associated locus, vgrG3. Here we present evidence to show how the host immune response, namely metabolic changes in response to infection may be exploited to support the organism’s adaptation to the lung microenvironment. In the evaluation of such a phenotypic response notable in chronic infections, the Type VI Secretion System (T6SS) of P. aeruginosa is selectively regulated by a host-specific metabolic product, itaconate. While P. aeruginosa contains genetic clusters for three (H1-, H2-, and H3-T6SS) evolutionarily distinct T6SSs, we found the H3-T6SS to be up-regulated significantly (p<0.05) in the presence of this anti-inflammatory signal. Characterization of this response reveals that itaconate induces metabolic stress in P. aeruginosa. In an acute pneumoniae mouse model, deletion of the H3-T6SS locus results in increased colonization of the murine lung. Analysis of bronchoalveolar lavage fluid from wild type and H3-T6SS null-infected mice reveals alterations in metabolic pathways including purine metabolism, carbon metabolism, and arginine biosynthesis. Overall our work outlines the H3-T6SS as a phenotypic response to metabolic stress induced by the host immune response, serving to mediate pathways essential in pathogenesis. Further understanding of such phenotypes as the T6SS implicated in chronic infection is essential in treatment interventions in the clinic.

Indiana University-Purdue University Indianapolis (IUPUI) / Pseudomonas aeruginosa is a gram negative opportunistic pathogen with the capacity to cause serious disease by forming biofilms, most notably in the lungs of cystic fibrosis (CF) patients. Biofilms are communities of microorganisms that adhere to a solid surface, undergo global regulatory changes, secrete exopolysaccharides, and are innately antibiotic resistant. Virulence modulation is an important tool utilized by P. aeruginosa to propagate infection and biofilm formation in the CF airway. Many different virulence modulatory pathways and proteins have been identified including the protein, MgtE. MgtE has recently been discovered and has been implicated in virulence modulation, as an isogeneic mutation of mgtE leads to increased cytotoxicity. To further elucidate the role of MgtE in P. aerugionsa infections, transcriptional and translational regulation of this protein following antibiotic treatment has been explored. I have demonstrated that mgtE is transcriptionally upregulated following antibiotic treatment of most of the twelve antibiotics tested utilizing RT-PCR and QRT-PCR. A novel model system was employed, which utilizes cystic fibrosis bronchial epithelial (CFBE) cells homozygous for the ΔF508 mutation for these studies. This model system allows P. aeruginosa biofilms to form on CFBE cells modeling the P. aeruginosa in the CF airway. Translational effects of antibiotic treatment on MgtE have been attempted via Western blotting and cytotoxicity assays. Furthermore, to explore the possibility that mgtE is interacting with a known regulatory pathway, a transposon-mutant library was utilized and the regulatory proteins, AlgR and NarX, among others have been identified as possibly interacting with MgtE. Lastly, an MgtE homologue from Staphylococcus aureus was utilized to further demonstrate the virulence modulatory effects of MgtE by demonstrating the expression of the homologue results in decreased cytotoxicity, exactly like expression of the native P. aeruginosa MgtE. This research explores a newly discovered protein that impacts cytotoxicity and biofilm formation and provides valuable information about P. aeruginosa virulence.

Indiana University-Purdue University Indianapolis (IUPUI) / The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen which readily establishes itself in the lungs of people with cystic fibrosis (CF). Most CF patients have life-long P. aeruginosa infections. By modulating its own virulence and forming biofilms, P. aeruginosa is able to evade both host immune responses and antibiotic treatments. Previous studies have shown that the magnesium transporter MgtE plays a role in virulence modulation by inhibiting transcription of the type III secretion system, a mechanism by which bacteria inject toxins directly into the eukaryotic host cell. MgtE had already been identified as a magnesium transporter, and thus its role in regulating cytotoxicity was indicative of dual functions for this protein. This research focused on a structure-function analysis of MgtE, with the hypothesis that the magnesium transport and cytotoxicity functions could be exerted independently. Cytotoxicity assays were conducted using a co-culture model system of cystic fibrosis bronchial epithelial cells and a ∆mgtE strain of P. aeruginosa transformed with plasmids carrying wild type or mutated mgtE. Magnesium transport was assessed using the same mgtE plasmids in a Salmonella strain deficient in all magnesium transporters. Through analysis of a number of mgtE mutants, we found two constructs – a mutation in a putative magnesium binding site, and an N-terminal truncation – which demonstrated a separation of functions. We further demonstrated the uncoupling of functions by showing that different mgtE mutants vary widely in their ability to regulate cytotoxicity, whether or not they are able to transport magnesium. Overall, these results support the hypothesis of MgtE as a dual function protein and may lead to a better understanding of the mechanisms underlying P. aeruginosa virulence. By understanding virulence mechanisms, we may be able to develop treatments to reduce infections and pave the way to better health for people with cystic fibrosis.

P. aeruginosa é um importante agente de infecções relacionadas à assistência em saúde. Habitualmente, o estabelecimento de infecções agudas é precedido pela colonização das mucosas dos pacientes. Não se sabe, porém, se os processos infecciosos são causados pelas próprias cepas bacterianas colonizadoras ou por outras com que os pacientes entrem em contato, dotadas ou não de maior potencial de virulência ou de resistência a antimicrobianos que as tornem mais eficientes como agentes infecciosos. Assim, este estudo teve como objetivos i) investigar a existência de potenciais diferenças entre amostras de P. aeruginosa que causaram apenas colonização e aquelas responsáveis por infecção, isoladas de um mesmo paciente, quanto a seus fenótipos de virulência e de não susceptibilidade a antimicrobiamos; ii) pesquisar a existência de associação entre características dos paciente, incluindo o tipo de evolução clínica, com as demais variáveis estudadas. No estudo foram incluídos 21 pacientes que desenvolveram infecção por P. aeruginosa durante sua internação no Centro de Terapia Intensiva do Hospital Universitário Clementino Fraga Filho, entre abril de 2007 e abril de 2008. De cada paciente foram selecionadas duas amostras bacterianas: a primeira isolada durante o episódio de infecção e a amostra colonizadora obtida imediatamente antes da ocorrência da infecção. As amostras selecionadas foram estudadas quanto a i) expressão de três mecanismos de virulência (citotoxicidade, aderência a células epiteliais respiratórias humanas e capacidade de formação de biofilme); ii) presença de genes codificadores das proteínas efetoras do sistema de secreção do tipo 3 (SST3 - exoS, exoT, exoU e exoY); iii) perfil de susceptibilidade a antimicrobianos, iv) perfil de fragmentação do DNA cromossômico por eletroforese em gel de campo pulsado (PFGE). As amostras bacterianas obtidas de infecções agudas foram significativamente mais citotóxicas que aquelas obtidas de colonização. Embora sem diferença estatística, a citotoxicidade das amostras que causaram infecção em pacientes que evoluíram para óbito foi superior à citotoxicidade das amostras de pacientes que sobreviveram. O gene que codifica a toxina ExoU foi detectado em 16 amostras (38%), sendo nove de colonização e sete de infecção. Não houve diferença significativa entre as amostras de colonização e infecção quanto à aderência, produção de biofilme, expressão dos genes do SST3 e não-susceptibilidade às diferentes classes de antimicrobianos. Também não foi encontrada associação entre a não-susceptibilidade à quinolona, ou a outras classes de antimicrobianos, e a presença do gene exoU. As 42 amostras de P. aeruginosa estudadas foram incluídas em 20 genótipos. Em 10 deles foi detectado o gene exoU. Amostras de um mesmo genótipo foram uniformes quanto à expressão dos genes do SST3 e a não-susceptibilidade aos antimicrobianos, mas não quanto às outras variáveis estudadas. Em apenas sete pacientes (33,3%), as amostras de colonização e de infecção pertenciam ao mesmo genótipo. Assim, nesse estudo, o estabelecimento do processo infeccioso resultou não da perda do equilíbrio estabelecido entre os mecanismos de agressão das amostras colonizadoras e os de defesa do hospedeiro e sim da introdução de nova cepa bacteriana no organismo hospedeiro, cepa esta dotada de maior potencial citotóxico. / P. aeruginosa is an important agent of healthcare-associated infections. The establishment of acute infectious episodes is usually preceded by colonization of patient mucosa. However, it remains unknown whether the infectious processes are caused by bacterial strains previously colonizing the patient or by additional strains the patient may come into contact. These new isolates may carry greater virulence potential or antibiotic resistance that makes them more efficient as an infecting agent. Thus, the objetives of the present study were i) to investigate the existence of potential differences between P. aeruginosa isolates obtained from a colonized mucosa and isolates accounting for infectious processes, recovered from the same patient, with respect to virulence phenotypes and non-susceptibility to antimicrobial agents; ii) to investigate the existence of association between patient features, including the type of clinical outcome, with bacterial characteristics. The study included 21 patients who developed P. aeruginosa infection during their stay in the Intensive Care Unit of the University Hospital Clementino Fraga Filho, from April 2007 to April 2008. Two P. aeruginosa isolates were selected from each patient: the first isolate recovered from the infectious episode and the colonizing isolate obtained immediately before the onset of the infection. Features from the isolates investigated included: i) expression of three virulence mechanisms (cytotoxicity, adherence to human respiratory epithelial cells and biofilm formation); ii) presence of the genes encoding type III secretion system effector proteins (TTSS, exoS , exoT , exoU and exoY); iii) antimicrobial susceptibility profile; iv) profile of the bacterial chromossomic DNA fragmentation following analysis by pulsed-field gel electrophoresis (PFGE). The bacterial isolates obtained from acute infections were significantly more cytotoxic than colonizing strains. Moreover, bacteria accounting for infectious episodes in patients who died were more cytotoxic than those recovered from patients who survived, although the differences were not statistically significant. The ExoU toxin encoding gene was detected in 16 (38%) P. aeruginosa isolates: nine colonizing and seven infecting strains. There was no significant difference between colonizing and infecting samples in their adherence, biofilm production, expression of TTSS genes and non- susceptibility to different classes of antimicrobials. There was also no association between non-susceptibility to quinolone, or to any other class of antimicrobial agents, and the presence of the exoU gene. Twenty PFGE genotypes were identified. Isolates from 10 genotypes harboured the exoU gene. Isolates included in the same PFGE genotype exhibited a similar profile of TTSS genes and non-susceptibility to antimicrobials, but not always a similar profile of expression the other variables investigated. In only seven patients (33.3%), the colonizing and infecting isolates belonged to a same genotype. Thus, in this study, the establishment of the infectious process did not result from the loss of the equilibrium established between the aggression mechanisms of colonizing bacteria and host defense but rather from the introduction, in the host organism, of a new bacterial strain, endowed with a greater cytotoxic potential.

P. aeruginosa é um importante agente de infecções relacionadas à assistência em saúde. Habitualmente, o estabelecimento de infecções agudas é precedido pela colonização das mucosas dos pacientes. Não se sabe, porém, se os processos infecciosos são causados pelas próprias cepas bacterianas colonizadoras ou por outras com que os pacientes entrem em contato, dotadas ou não de maior potencial de virulência ou de resistência a antimicrobianos que as tornem mais eficientes como agentes infecciosos. Assim, este estudo teve como objetivos i) investigar a existência de potenciais diferenças entre amostras de P. aeruginosa que causaram apenas colonização e aquelas responsáveis por infecção, isoladas de um mesmo paciente, quanto a seus fenótipos de virulência e de não susceptibilidade a antimicrobiamos; ii) pesquisar a existência de associação entre características dos paciente, incluindo o tipo de evolução clínica, com as demais variáveis estudadas. No estudo foram incluídos 21 pacientes que desenvolveram infecção por P. aeruginosa durante sua internação no Centro de Terapia Intensiva do Hospital Universitário Clementino Fraga Filho, entre abril de 2007 e abril de 2008. De cada paciente foram selecionadas duas amostras bacterianas: a primeira isolada durante o episódio de infecção e a amostra colonizadora obtida imediatamente antes da ocorrência da infecção. As amostras selecionadas foram estudadas quanto a i) expressão de três mecanismos de virulência (citotoxicidade, aderência a células epiteliais respiratórias humanas e capacidade de formação de biofilme); ii) presença de genes codificadores das proteínas efetoras do sistema de secreção do tipo 3 (SST3 - exoS, exoT, exoU e exoY); iii) perfil de susceptibilidade a antimicrobianos, iv) perfil de fragmentação do DNA cromossômico por eletroforese em gel de campo pulsado (PFGE). As amostras bacterianas obtidas de infecções agudas foram significativamente mais citotóxicas que aquelas obtidas de colonização. Embora sem diferença estatística, a citotoxicidade das amostras que causaram infecção em pacientes que evoluíram para óbito foi superior à citotoxicidade das amostras de pacientes que sobreviveram. O gene que codifica a toxina ExoU foi detectado em 16 amostras (38%), sendo nove de colonização e sete de infecção. Não houve diferença significativa entre as amostras de colonização e infecção quanto à aderência, produção de biofilme, expressão dos genes do SST3 e não-susceptibilidade às diferentes classes de antimicrobianos. Também não foi encontrada associação entre a não-susceptibilidade à quinolona, ou a outras classes de antimicrobianos, e a presença do gene exoU. As 42 amostras de P. aeruginosa estudadas foram incluídas em 20 genótipos. Em 10 deles foi detectado o gene exoU. Amostras de um mesmo genótipo foram uniformes quanto à expressão dos genes do SST3 e a não-susceptibilidade aos antimicrobianos, mas não quanto às outras variáveis estudadas. Em apenas sete pacientes (33,3%), as amostras de colonização e de infecção pertenciam ao mesmo genótipo. Assim, nesse estudo, o estabelecimento do processo infeccioso resultou não da perda do equilíbrio estabelecido entre os mecanismos de agressão das amostras colonizadoras e os de defesa do hospedeiro e sim da introdução de nova cepa bacteriana no organismo hospedeiro, cepa esta dotada de maior potencial citotóxico. / P. aeruginosa is an important agent of healthcare-associated infections. The establishment of acute infectious episodes is usually preceded by colonization of patient mucosa. However, it remains unknown whether the infectious processes are caused by bacterial strains previously colonizing the patient or by additional strains the patient may come into contact. These new isolates may carry greater virulence potential or antibiotic resistance that makes them more efficient as an infecting agent. Thus, the objetives of the present study were i) to investigate the existence of potential differences between P. aeruginosa isolates obtained from a colonized mucosa and isolates accounting for infectious processes, recovered from the same patient, with respect to virulence phenotypes and non-susceptibility to antimicrobial agents; ii) to investigate the existence of association between patient features, including the type of clinical outcome, with bacterial characteristics. The study included 21 patients who developed P. aeruginosa infection during their stay in the Intensive Care Unit of the University Hospital Clementino Fraga Filho, from April 2007 to April 2008. Two P. aeruginosa isolates were selected from each patient: the first isolate recovered from the infectious episode and the colonizing isolate obtained immediately before the onset of the infection. Features from the isolates investigated included: i) expression of three virulence mechanisms (cytotoxicity, adherence to human respiratory epithelial cells and biofilm formation); ii) presence of the genes encoding type III secretion system effector proteins (TTSS, exoS , exoT , exoU and exoY); iii) antimicrobial susceptibility profile; iv) profile of the bacterial chromossomic DNA fragmentation following analysis by pulsed-field gel electrophoresis (PFGE). The bacterial isolates obtained from acute infections were significantly more cytotoxic than colonizing strains. Moreover, bacteria accounting for infectious episodes in patients who died were more cytotoxic than those recovered from patients who survived, although the differences were not statistically significant. The ExoU toxin encoding gene was detected in 16 (38%) P. aeruginosa isolates: nine colonizing and seven infecting strains. There was no significant difference between colonizing and infecting samples in their adherence, biofilm production, expression of TTSS genes and non- susceptibility to different classes of antimicrobials. There was also no association between non-susceptibility to quinolone, or to any other class of antimicrobial agents, and the presence of the exoU gene. Twenty PFGE genotypes were identified. Isolates from 10 genotypes harboured the exoU gene. Isolates included in the same PFGE genotype exhibited a similar profile of TTSS genes and non-susceptibility to antimicrobials, but not always a similar profile of expression the other variables investigated. In only seven patients (33.3%), the colonizing and infecting isolates belonged to a same genotype. Thus, in this study, the establishment of the infectious process did not result from the loss of the equilibrium established between the aggression mechanisms of colonizing bacteria and host defense but rather from the introduction, in the host organism, of a new bacterial strain, endowed with a greater cytotoxic potential.

Indiana University-Purdue University Indianapolis (IUPUI) / The patients with Cystic fibrosis (CF), an inherent genetic disorder, suffer from chronic bacterial infection in the lung. In CF, modification of epithelial cells leads to alteration of the lung environment, such as inhibition of ciliary bacterial clearance and accumulation of thickened mucus in the airways. Exploiting these conditions, opportunistic pathogens like Pseudomonas aeruginosa cause lifelong persistent infection in the CF lung by forming into antibiotic-resistant aggregated communities called biofilms. Airway infections as well as inflammation are the two major presentations of CF lung disease. P. aeruginosa strains isolated from CF lungs often contain mutations in the mucA gene, and this mutation results in higher level expression of bacterial polysaccharides and toxic lipoproteins. In a previous work, we have found a putative lipoprotein gene (PA4326) which is overexpressed in antibiotic-induced biofilm formed on cultured CF-derived airway cells. In the current work, we speculated that this particular putative lipoprotein affects cellular cytotoxicity and immune-stimulation in the epithelial cells. We found that mutation of this gene (ΔPA4326) results in reduced airway cell killing without affecting other common virulence factors.Moreover, we observed that this gene was able to stimulate secretion of the proinflammatory cytokine IL-8 from host cells. Interestingly, we also found that ΔPA4326 mutant strains produced less pyocyanin exotoxin compared to the wild type. Furthermore, our results suggest that PA4326 regulates expression of the pyocyanin biosynthesis gene phzM, leading to the reduced pyocyanin phenotype. Overall, these findings implicate PA4326 as a virulence factor in Pseudomonas aeruginosa. In the future, understating the molecular interplay between the epithelial cells and putative lipoproteins like PA4326 may lead to development of novel anti-inflammatory therapies that would lessen the suffering of CF patients.