RESUMO
Investigation of a dengue case in a laboratory worker in North Carolina, USA, revealed that the case-patient prepared high-titer dengue virus stocks soon before illness onset. Improper doffing of gloves with an open finger wound likely resulted in cutaneous exposure. This case reinforces recommendations for enhanced precautions when working with high-titer dengue virus.
Assuntos
Vírus da Dengue , Dengue , Dengue/diagnóstico , Dengue/epidemiologia , Vírus da Dengue/genética , Humanos , Laboratórios , North Carolina/epidemiologia , Estados Unidos/epidemiologiaRESUMO
The Mycobacterium tuberculosis mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel M. tuberculosis growth inhibitors for differential growth inhibition of wild-type M. tuberculosis compared to the growth of a pool of 24 unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular M. tuberculosisM. tuberculosis cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species M. abscessus Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Benzamidas/farmacologia , Benzotiazóis/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Piridinas/farmacologia , Antituberculosos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Benzamidas/síntese química , Benzotiazóis/síntese química , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Fatores Corda/antagonistas & inibidores , Fatores Corda/biossíntese , Fatores Corda/metabolismo , Farmacorresistência Bacteriana/genética , Galactanos/metabolismo , Expressão Gênica , Ensaios de Triagem em Larga Escala , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/genética , Mycobacterium abscessus/crescimento & desenvolvimento , Mycobacterium abscessus/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Piridinas/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequenciamento Completo do GenomaRESUMO
Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is a deadly disease that requires a long course of treatment. The emergence of drug-resistant strains has driven efforts to discover new small molecules that can kill the bacterium. Here, we report characterizations of the compound HC2091, which kills M. tuberculosis in a time- and dose-dependent manner in vitro and inhibits M. tuberculosis growth in macrophages. Whole-genome sequencing of spontaneous HC2091-resistant mutants identified single-nucleotide variants in the mmpL3 mycolic acid transporter gene. HC2091-resistant mutants do not exhibit cross-resistance with the well-characterized Mycobacterium membrane protein large 3 (MmpL3) inhibitor SQ109, suggesting a distinct mechanism of interaction with MmpL3. Additionally, HC2091 does not modulate bacterial membrane potential or kill nonreplicating M. tuberculosis, thus acting differently from other known MmpL3 inhibitors. RNA sequencing (RNA-seq) transcriptional profiling and lipid profiling of M. tuberculosis treated with HC2091 or SQ109 show that the two compounds target a similar pathway. HC2091 has a chemical structure dissimilar to those of previously described MmpL3 inhibitors, supporting the notion that HC2091 is a new class of MmpL3 inhibitor.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Ácidos Micólicos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Tuberculose/genética , Tuberculose/metabolismo , Tuberculose/microbiologiaRESUMO
Rhodococcus equi is a facultative intracellular pathogen of macrophages, relying on the presence of a conjugative virulence plasmid harboring a 21-kb pathogenicity island (PAI) for growth in host macrophages. The PAI encodes a family of 6 virulence-associated proteins (Vaps) in addition to 20 other proteins. The contribution of these to virulence has remained unclear. We show that the presence of only 3 virulence plasmid genes (of 73 in total) is required and sufficient for intracellular growth. These include a single vap family member, vapA, and two PAI-located transcriptional regulators, virR and virS. Both transcriptional regulators are essential for wild-type-level expression of vapA, yet vapA expression alone is not sufficient to allow intracellular growth. A whole-genome microarray analysis revealed that VirR and VirS substantially integrate themselves into the chromosomal regulatory network, significantly altering the transcription of 18% of all chromosomal genes. This pathoadaptation involved significant enrichment of select gene ontologies, in particular, enrichment of genes involved in transport processes, energy production, and cellular metabolism, suggesting a major change in cell physiology allowing the bacterium to grow in the hostile environment of the host cell. The results suggest that following the acquisition of the virulence plasmid by an avirulent ancestor of R. equi, coevolution between the plasmid and the chromosome took place, allowing VirR and VirS to regulate the transcription of chromosomal genes in a process that ultimately promoted intracellular growth. Our findings suggest a mechanism for cooption of existing chromosomal traits during the evolution of a pathogenic bacterium from an avirulent saprophyte.
Assuntos
Infecções por Actinomycetales/microbiologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Macrófagos/microbiologia , Plasmídeos/genética , Rhodococcus equi/fisiologia , Transcriptoma , Adaptação Fisiológica , Animais , Proteínas de Bactérias/metabolismo , Humanos , Camundongos , Plasmídeos/metabolismo , Rhodococcus equi/genética , Rhodococcus equi/crescimento & desenvolvimento , Transcrição Gênica , Fatores de Virulência/genéticaRESUMO
Virulence of the intracellular pathogen Rhodococcus equi depends on a 21.3-kb pathogenicity island located on a conjugative plasmid. To date, the only nonregulatory pathogenicity island-encoded virulence factor identified is the cell envelope-associated VapA protein. Although the pathogenicity islands from porcine and equine R. equi isolates have undergone major rearrangements, the virR operon (virR-icgA-vapH-orf7-virS) is highly conserved in both, suggesting these genes play an important role in pathogenicity. VirR and VirS are transcriptional regulators controlling expression of pathogenicity island genes, including vapA. Here, we show that while vapH and orf7 are dispensable for intracellular growth of R. equi, deletion of icgA, formerly known as orf5, encoding a major facilitator superfamily transport protein, elicited an enhanced growth phenotype in macrophages and a significant reduction in macrophage viability, while extracellular growth in broth remained unaffected. Transcription of virS, located downstream of icgA, and vapA was not affected by the icgA deletion during growth in broth or in macrophages, showing that the enhanced growth phenotype caused by deletion of icgA was not mediated through abnormal transcription of these genes. Transcription of icgA increased 6-fold within 2 h following infection of macrophages and remained significantly higher 48 h postinfection compared to levels at the start of the infection. The major facilitator superfamily transport protein IcgA is the first factor identified in R. equi that negatively affects intracellular replication. Aside from VapA, it is only the second pathogenicity island-encoded structural protein shown to play a direct role in intracellular growth of this pathogenic actinomycete.
Assuntos
Proteínas de Bactérias/metabolismo , Rhodococcus equi/metabolismo , Rhodococcus equi/fisiologia , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Regulação Bacteriana da Expressão Gênica/fisiologia , Macrófagos/microbiologia , Camundongos , Transcriptoma , Virulência , Fatores de Virulência/genéticaRESUMO
We previously showed that the facultative intracellular pathogen Rhodococcus equi produces a nondiffusible and catecholate-containing siderophore (rhequibactin) involved in iron acquisition during saprophytic growth. Here, we provide evidence that the rhbABCDE cluster directs the biosynthesis of a hydroxamate siderophore, rhequichelin, that plays a key role in virulence. The rhbC gene encodes a nonribosomal peptide synthetase that is predicted to produce a tetrapeptide consisting of N(5)-formyl-N(5)-hydroxyornithine, serine, N(5)-hydroxyornithine, and N(5)-acyl-N(5)-hydroxyornithine. The other rhb genes encode putative tailoring enzymes mediating modification of ornithine residues incorporated into the hydroxamate product of RhbC. Transcription of rhbC was upregulated during growth in iron-depleted medium, suggesting that it plays a role in iron acquisition. This was confirmed by deletion of rhbCD, rendering the resulting strain R. equi SID2 unable to grow in the presence of the iron chelator 2,2-dipyridyl. Supernatant of the wild-type strain rescued the phenotype of R. equi SID2. The importance of rhequichelin in virulence was highlighted by the rapid increase in transcription levels of rhbC following infection and the inability of R. equi SID2 to grow within macrophages. Unlike the wild-type strain, R. equi SID2 was unable to replicate in vivo and was rapidly cleared from the lungs of infected mice. Rhequichelin is thus a key virulence-associated factor, although nonpathogenic Rhodococcus species also appear to produce rhequichelin or a structurally closely related compound. Rhequichelin biosynthesis may therefore be considered an example of cooption of a core actinobacterial trait in the evolution of R. equi virulence.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ácidos Hidroxâmicos/metabolismo , Ferro/metabolismo , Oligopeptídeos/metabolismo , Rhodococcus equi/patogenicidade , Sideróforos/metabolismo , Fatores de Virulência/metabolismo , Animais , Células Cultivadas , Feminino , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Rhodococcus equi/genética , Rhodococcus equi/crescimento & desenvolvimento , Rhodococcus equi/metabolismo , Virulência , Fatores de Virulência/genéticaRESUMO
Mycobacterium tuberculosis (Mtb) senses and adapts to host immune cues as part of its pathogenesis. One environmental cue sensed by Mtb is the acidic pH of its host niche in the macrophage phagosome. Disrupting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. Previously, a high throughput screen of a â¼220 000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. The screen discovered chemical probes that kill Mtb at pH 5.7 but are inactive at pH 7.0. In this study, AC2P20 was prioritized for continued study to test the hypothesis that it was targeting Mtb pathways associated with pH-driven adaptation. RNAseq transcriptional profiling studies showed AC2P20 modulates expression of genes associated with redox homeostasis. Gene enrichment analysis revealed that the AC2P20 transcriptional profile had significant overlap with a previously characterized pH-selective inhibitor, AC2P36. Like AC2P36, we show that AC2P20 kills Mtb by selectively depleting free thiols at acidic pH. Mass spectrometry studies show the formation of a disulfide bond between AC2P20 and reduced glutathione, supporting a mechanism where AC2P20 is able to deplete intracellular thiols and dysregulate redox homeostasis. The observation of two independent molecules targeting free thiols to kill Mtb at acidic pH further supports that Mtb has restricted redox homeostasis and sensitivity to thiol-oxidative stress at acidic pH.
RESUMO
Rhodococcus equi, a facultative intracellular pathogen of macrophages, causes severe, life-threatening pneumonia in young foals and in people with underlying immune deficiencies. R. equi virulence is dependent on the presence of a large virulence plasmid that houses a pathogenicity island (PAI) encoding a novel family of surface-localized and secreted proteins of largely unknown function termed the virulence-associated proteins (VapACDEFGHI). To date, vapA and its positive regulators virR and orf8 are the only experimentally established virulence genes residing on the virulence plasmid. In this study, a PAI deletion mutant was constructed and, as anticipated, was attenuated for growth both in macrophages and in mice due to the absence of vapA expression. Expression of vapA in the PAI mutant from a constitutive promoter, thereby eliminating the requirement for the PAI-encoded vapA regulators, resulted in delayed bacterial clearance in vivo, yet full virulence was not restored, indicating that additional virulence genes are indeed located within the deleted pathogenicity island region. Based on previous reports demonstrating that the PAI-carried gene vapG is highly upregulated in macrophages and in the lungs of R. equi-infected foals, we hypothesized that vapG could be an important virulence factor. However, analysis of a marked vapG deletion mutant determined the gene to be dispensable for growth in macrophages and in vivo in mice.
Assuntos
Proteínas de Bactérias/fisiologia , Ilhas Genômicas , Rhodococcus equi/genética , Rhodococcus equi/patogenicidade , Fatores de Virulência/fisiologia , Infecções por Actinomycetales/microbiologia , Infecções por Actinomycetales/patologia , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Feminino , Fígado/microbiologia , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos , Camundongos SCID , Plasmídeos , Deleção de Sequência , Baço/microbiologia , Virulência , Fatores de Virulência/genéticaRESUMO
The proportion of meningococcal disease in the United States, South Africa, and Israel caused by Neisseria meningitidis serogroup Y (NmY) was greater than the worldwide average during the period 1999-2002. Genotypic characterization of 300 NmY isolates by multilocus sequence typing, 16S rRNA gene sequencing, and PorA variable region typing was conducted to determine the relationships of the isolates from these three countries. Seventy different genotypes were found. Two groups of ST-23 clonal complex isolates accounted for 88% of the U.S. isolates, 12% of the South African isolates, and 96% of the isolates from Israel. The single common clone (ST-23/16S-19/P1.5-2,10-1) represented 57, 5, and 35% of the NmY isolates from the United States, South Africa, and Israel. The predominant clone in South Africa (ST-175/16S-21/P1.5-1,2-2), and 11 other closely related clones made up 77% of the South African study isolates and were not found among the isolates from the United States or Israel. ST-175 was the predicted founder of the ST-175 clonal complex, and isolates of ST-175 and related sequence types have been described previously in other African countries. Continued active surveillance and genetic characterization of NmY isolates causing disease in the United States, South Africa, and Israel will provide valuable data for local and global epidemiology and allow monitoring for any expansion of existing clonal complexes and detection of the emergence of new virulent clones in the population.
Assuntos
Técnicas de Tipagem Bacteriana , Infecções Meningocócicas/microbiologia , Neisseria meningitidis Sorogrupo Y/genética , Neisseria meningitidis Sorogrupo Y/isolamento & purificação , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genótipo , Humanos , Israel , Epidemiologia Molecular , Filogenia , Porinas/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência , África do Sul , Estados UnidosRESUMO
BACKGROUND: In the African meningitis belt, Neisseria meningitidis serogroup W135 has emerged as a cause of epidemic disease. The establishment of W135 as the predominant cause of endemic disease has not been described. METHODS: We conducted national laboratory-based surveillance for invasive meningococcal disease during 2000-2005. The system was enhanced in 2003 to include clinical data collection of cases from sentinel sites. Isolates were characterized by pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS: A total of 2135 cases of invasive meningococcal disease were reported, of which 1113 (52%) occurred in Gauteng Province, South Africa. In this province, rates of disease increased from 0.8 cases per 100,000 persons in 2000 to 4.0 cases per 100,000 persons in 2005; the percentage due to serogroup W135 increased from 7% (4 of 54 cases) to 75% (221 of 295 cases). The median age of patients infected with serogroup W135 was 5 years (interquartile range, 2-23 years), compared with 21 years (range, 8-26 years) for those infected with serogroup A (P<.001). The incidence of W135 disease increased in all age groups. Rates were highest among infants (age, <1 year), increasing from 5.1 cases per 100,000 persons in 2003 to 21.5 cases per 100,000 persons in 2005. Overall case-fatality rates doubled, from 11% in 2003 to 22% in 2005. Serogroup W135 was more likely to cause meningococcemia than was serogroup A (82 [28%] of 297 cases vs. 11 [8%] of 141 cases; odds ratio, 8.9, 95% confidence interval, 2.2-36.3). A total of 285 (95%) of 301 serogroup W135 isolates were identified as 1 clone by pulsed-field gel electrophoresis; 7 representative strains belonged to the ST-11/ET-37 complex. CONCLUSIONS: Serogroup W135 has become endemic in Gauteng, South Africa, causing disease of greater severity than did the previous predominant serogroup A strain.
Assuntos
Doenças Transmissíveis Emergentes/microbiologia , Doenças Endêmicas , Meningite Meningocócica/epidemiologia , Neisseria meningitidis Sorogrupo W-135/isolamento & purificação , Adolescente , Adulto , Criança , Pré-Escolar , Doenças Transmissíveis Emergentes/epidemiologia , Eletroforese em Gel de Campo Pulsado/métodos , Feminino , Humanos , Lactente , Masculino , Meningite Meningocócica/microbiologia , Meningite Meningocócica/mortalidade , Pessoa de Meia-Idade , Neisseria meningitidis Sorogrupo W-135/genética , Vigilância de Evento Sentinela , África do Sul/epidemiologiaRESUMO
Rhodococcus equi is a facultative intracellular bacterium of macrophages and is an important pathogen of animals and immunocompromised people wherein disease results in abcessation of the lungs and other sites. Prior work has shown that the presence of the major virulence determinant, VapA, encoded on the pVAPA-type plasmid, disrupts normal phagosome development and is essential for bacterial replication within macrophages. pVAPA- type plasmids are typical of R. equi strains derived from foals while strains from pigs carry plasmids of the pVAPB-type, lacking vapA, and those from humans harbor various types of plasmids including pVAPA and pVAPB. Through the creation and analysis of a series of gene deletion mutants, we found that vapK1 or vapK2 is required for optimal intracellular replication of an R. equi isolate carrying a pVAPB plasmid type. Complementation analysis of a ΔvapA R. equi strain with vapK1 or vapK2 showed the VapK proteins of the pVAPB-type plasmid could restore replication capacity to the macrophage growth-attenuated ΔvapA strain. Additionally, in contrast to the intracellular growth capabilities displayed by an equine R. equi transconjugant strain carrying a pVAPB-type plasmid, a transconjugant strain carrying a pVAPB-type plasmid deleted of vapK1 and vapK2 proved incapable of replication in equine macrophages. Cumulatively, these data indicate that VapK1 and K2 are functionally equivalent to VapA.
Assuntos
Proteínas de Bactérias/genética , Macrófagos/microbiologia , Plasmídeos , Rhodococcus equi/genética , Rhodococcus equi/patogenicidade , Fatores de Virulência/genética , Infecções por Actinomycetales/microbiologia , Infecções por Actinomycetales/veterinária , Animais , Células Cultivadas , Feminino , Doenças dos Cavalos/microbiologia , Cavalos , Camundongos Endogâmicos BALB C , Mutação , Rhodococcus equi/crescimento & desenvolvimento , Rhodococcus equi/isolamento & purificaçãoRESUMO
Mycobacterium tuberculosis (Mtb) must sense and adapt to immune pressures such as acidic pH during pathogenesis. The goal of this study was to isolate compounds that inhibit acidic pH resistance, thus defining virulence pathways that are vulnerable to chemotherapy. Here, we report that the compound AC2P36 selectively kills Mtb at acidic pH and potentiates the bactericidal activity of isoniazid, clofazimine, and diamide. We show that AC2P36 activity is associated with thiol stress and causes an enhanced accumulation of intracellular reactive oxygen species at acidic pH. Mechanism of action studies demonstrate that AC2P36 directly depletes Mtb thiol pools, with enhanced depletion of free thiols at acidic pH. These findings support that Mtb is especially vulnerable to thiol stress at acidic pH and that chemical depletion of thiol pools is a promising target to promote Mtb killing and potentiation of antimicrobials.
Assuntos
Antibacterianos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Pirimidinas/farmacologia , Compostos de Sulfidrila/metabolismo , Sulfonas/farmacologia , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glutationa/química , Concentração de Íons de Hidrogênio , Mycobacterium tuberculosis/crescimento & desenvolvimento , Estresse Oxidativo/efeitos dos fármacos , Pirimidinas/química , Espécies Reativas de Oxigênio/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/química , Sulfonas/químicaRESUMO
We describe the epidemiology of invasive meningococcal disease in South Africa from August 1999 through July 2002, as reported to a laboratory-based surveillance system. Neisseria meningitidis isolates were further characterized. In total, 854 cases of laboratory-confirmed disease were reported, with an annual incidence rate of 0.64/100,000 population. Incidence was highest in infants < 1 year of age. Serogroup B caused 41% of cases; serogroup A, 23%; serogroup Y, 21%; serogroup C, 8%; and serogroup W135, 5%. Serogroup B was the predominant serogroup in Western Cape Province, and disease rates remained stable. Serogroup A was most prevalent in Gauteng Province and increased over the 3 years. On pulsed-field gel electrophoresis analysis, serogroup A strains showed clonality, and serogroup B demonstrated considerable diversity. Selected isolates of serogroup A belonged to sequence type (ST)-1 (subgroup I/II) complex, serogroup B to ST-32/electrophoretic type (ET)-5 complex, and serogroup W135 to ST-11/ET-37 complex.
Assuntos
Infecções Meningocócicas/epidemiologia , Humanos , Infecções Meningocócicas/microbiologia , Epidemiologia Molecular , Neisseria meningitidis/classificação , Sorotipagem , África do Sul/epidemiologiaRESUMO
We characterized five Neisseria meningitidis serogroup C isolates from a Chicago outbreak of meningococcal disease that occurred in 2003 among a community of men who have sex with men. Isolates from this outbreak were identical to each other but distinct from the clone that caused a similar outbreak in Canada in 2001.