RESUMEN
Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS), or "visceral myopathy," is a severe early onset disorder characterized by impaired muscle contractility in the bladder and intestines. Five genes are linked to MMIHS: primarily ACTG2, but also LMOD1, MYH11, MYLK, and MYL9. Here we describe a three-year-old girl with bilateral hydronephrosis diagnosed at 20 weeks gestation and congenital mydriasis (both of which have been previously observed among individuals with MMIHS). A clinical diagnosis of MMIHS was made based upon the presence of megacystis, lack of urinary bladder peristalsis, and intestinal pseudo-obstruction. After initial testing of ACTG2 was negative, further sequencing and deletion/duplication testing was performed on the LMOD1, MYH11,MYLK, and MYL9 genes. We identified two heterozygous loss of function variants in MYL9: an exon 4 deletion and a nine base pair deletion that removes the canonical splicing donor site at exon 2 (NM_006097.5:c.184+2_184+10del). Parental testing confirmed these variants to be in trans in our proband. To our knowledge, only one other individual with MMIHS has biallelic mutations in MYL9 (a homozygous deletion encompassing exon 4). We suggest MYL9 be targeted on genetic testing panels for MMIHS, smooth muscle myopathies, and cardiovascular phenotypes.
Asunto(s)
Anomalías Múltiples/genética , Colon/anomalías , Seudoobstrucción Intestinal/genética , Mutación con Pérdida de Función , Cadenas Ligeras de Miosina/genética , Vejiga Urinaria/anomalías , Anomalías Múltiples/patología , Preescolar , Colon/patología , Femenino , Heterocigoto , Humanos , Seudoobstrucción Intestinal/patología , Vejiga Urinaria/patologíaRESUMEN
Resistance to the first-line antituberculosis (TB) drug isoniazid (INH) is widespread, and the mechanism of resistance is unknown in approximately 15% of INH-resistant (INH-R) strains. To improve molecular detection of INH-R TB, we used whole-genome sequencing (WGS) to analyze 52 phenotypically INH-R Mycobacterium tuberculosis complex (MTBC) clinical isolates that lacked the common katG S315T or inhA promoter mutations. Approximately 94% (49/52) of strains had mutations at known INH-associated loci that were likely to confer INH resistance. All such mutations would be detectable by sequencing more DNA adjacent to existing target regions. Use of WGS minimized the chances of missing infrequent INH resistance mutations outside commonly targeted hotspots. We used recombineering to generate 12 observed clinical katG mutations in the pansusceptible H37Rv reference strain and determined their impact on INH resistance. Our functional genetic experiments have confirmed the role of seven suspected INH resistance mutations and discovered five novel INH resistance mutations. All recombineered katG mutations conferred resistance to INH at a MIC of ≥0.25 µg/ml and should be added to the list of INH resistance determinants targeted by molecular diagnostic assays. We conclude that WGS is a useful tool for detecting uncommon INH resistance mutations that would otherwise be missed by current targeted molecular testing methods and suggest that its use (or use of expanded conventional or next-generation-based targeted sequencing) may provide earlier diagnosis of INH-R TB.
Asunto(s)
Antituberculosos/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación/genética , Tuberculosis/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/genéticaRESUMEN
Neisseria gonorrhoeae produces two transferrin binding proteins, TbpA and TbpB, which together enable efficient iron transport from human transferrin. We demonstrate that expression of the tbp genes is controlled by MisR, a response regulator in the two-component regulatory system that also includes the sensor kinase MisS. The tbp genes were up-regulated in the misR mutant under iron-replete conditions but were conversely down-regulated in the misR mutant under iron-depleted conditions. The misR mutant was capable of transferrin-iron uptake at only 50% of wild-type levels, consistent with decreased tbp expression. We demonstrate that phosphorylated MisR specifically binds to the tbpBA promoter and that MisR interacts with five regions upstream of the tbpB start codon. These analyses confirm that MisR directly regulates tbpBA expression. The MisR binding sites in the gonococcus are only partially conserved in Neisseria meningitidis, which may explain why tbpBA was not MisR-regulated in previous studies using this related pathogen. This is the first report of a trans-acting protein factor other than Fur that can directly contribute to gonococcal tbpBA regulation.
Asunto(s)
Neisseria gonorrhoeae/genética , Proteína A de Unión a Transferrina/genética , Proteína B de Unión a Transferrina/genética , Sitios de Unión , Hierro/metabolismo , Neisseria gonorrhoeae/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transactivadores/metabolismo , Transferrina/metabolismo , Proteína A de Unión a Transferrina/metabolismo , Proteína B de Unión a Transferrina/metabolismoRESUMEN
During infection, the sexually transmitted pathogen Neisseria gonorrhoeae (the gonococcus) encounters numerous host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogen Neisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator but not the MisS sensor kinase is involved in constitutive and inducible CAMP resistance and is also required for intrinsic low-level resistance to aminoglycosides. The 4- to 8-fold increased susceptibility of misR-deficient gonococci to CAMPs and aminoglycosides was independent of phosphoethanolamine decoration of lipid A and the levels of the MtrCDE efflux pump and seemed to correlate with a general increase in membrane permeability. Transcriptional profiling and biochemical studies confirmed that expression of lptA and mtrCDE was not impacted by the loss of MisR. However, several genes encoding proteins involved in membrane integrity and redox control gave evidence of being MisR regulated. We propose that MisR modulates the levels of gonococcal susceptibility to antimicrobials by influencing the expression of genes involved in determining membrane integrity.
Asunto(s)
Aminoglicósidos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Bacterianas/metabolismo , Gonorrea/metabolismo , Neisseria gonorrhoeae/metabolismo , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Gonorrea/tratamiento farmacológico , Humanos , Lípido A/metabolismo , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria meningitidis/efectos de los fármacos , Neisseria meningitidis/metabolismoRESUMEN
Autophagy, an ancient homeostasis mechanism for macromolecule degradation, performs an important role in host defense by facilitating pathogen elimination. To counteract this host defense strategy, bacterial pathogens have evolved a variety of mechanisms to avoid or otherwise dysregulate autophagy by phagocytic cells so as to enhance their survival during infection. Neisseria gonorrhoeae is a strictly human pathogen that causes the sexually transmitted infection, gonorrhea. Phosphoethanolamine (PEA) addition to the 4' position of the lipid A (PEA-lipid A) moiety of the lipooligosaccharide (LOS) produced by gonococci performs a critical role in this pathogen's ability to evade innate defenses by conferring decreased susceptibility to cationic antimicrobial (or host-defense) peptides, complement-mediated killing by human serum and intraleukocytic killing by human neutrophils compared to strains lacking this PEA decoration. Heretofore, however, it was not known if gonococci can evade autophagy and if so, whether PEA-lipid A contributes to this ability. Accordingly, by using murine macrophages and human macrophage-like phagocytic cell lines we investigated if PEA decoration of gonococcal lipid A modulates autophagy formation. We report that infection with PEA-lipid A-producing gonococci significantly reduced autophagy flux in murine and human macrophages and enhanced gonococcal survival during their association with macrophages compared to a PEA-deficient lipid A mutant. Our results provide further evidence that PEA-lipid A produced by gonococci is a critical component in the ability of this human pathogen to evade host defenses.
Asunto(s)
Autofagia/fisiología , Etanolaminas , Lípido A/metabolismo , Macrófagos/microbiología , Neisseria gonorrhoeae/patogenicidad , Animales , Autofagia/efectos de los fármacos , Línea Celular/microbiología , Quimiocinas/metabolismo , Disacáridos/farmacología , Interacciones Huésped-Patógeno , Humanos , Lípido A/química , Ratones , Neisseria gonorrhoeae/metabolismo , Fagosomas/metabolismo , Fagosomas/microbiología , Fosfatos de Azúcar/farmacologíaRESUMEN
Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae. The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB. We localized the 5' endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Neisseria gonorrhoeae/genética , Secuencias Reguladoras de Ácido Ribonucleico/genética , Proteína A de Unión a Transferrina/genética , Proteína B de Unión a Transferrina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Genes Reporteros , Gonorrea/microbiología , Secuencias Invertidas Repetidas/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Neisseria gonorrhoeae/metabolismo , Operón/genética , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genética , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Análisis de Secuencia de ARN , Proteína A de Unión a Transferrina/metabolismo , Proteína B de Unión a Transferrina/metabolismoRESUMEN
Phosphoethanolamine (PEA) decoration of lipid A produced by Neisseria gonorrhoeae has been linked to bacterial resistance to cationic antimicrobial peptides/proteins (CAMPs) and in vivo fitness during experimental infection. We now report that the lptA gene, which encodes the PEA transferase responsible for this decoration, is in an operon and that high-frequency mutation in a polynucleotide repeat within lptA can influence gonococcal resistance to CAMPs.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Etanolaminofosfotransferasa/genética , Etanolaminas/química , Lípido A/química , Neisseria gonorrhoeae/efectos de los fármacos , Antibacterianos/farmacología , Secuencia de Bases , Farmacorresistencia Bacteriana/genética , Etanolaminofosfotransferasa/biosíntesis , Pruebas de Sensibilidad Microbiana , Neisseria gonorrhoeae/genéticaRESUMEN
Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted infection termed gonorrhea. The gonococcus can survive extracellularly and intracellularly, but in both environments the bacteria must acquire iron from host proteins for survival. However, upon infection the host uses a defensive response by limiting the bioavailability of iron by a number of mechanisms including the enhanced expression of hepcidin, the master iron-regulating hormone, which reduces iron uptake from the gut and retains iron in macrophages. The host also secretes the antibacterial protein NGAL, which sequesters bacterial siderophores and therefore inhibits bacterial growth. To learn whether intracellular gonococci can subvert this defensive response, we examined expression of host genes that encode proteins involved in modulating levels of intracellular iron. We found that N. gonorrhoeae can survive in association (tightly adherent and intracellular) with monocytes and macrophages and upregulates a panel of its iron-responsive genes in this environment. We also found that gonococcal infection of human monocytes or murine macrophages resulted in the upregulation of hepcidin, NGAL, and NRAMP1 as well as downregulation of the expression of the gene encoding the short chain 3-hydroxybutyrate dehydrogenase (BDH2); BDH2 catalyzes the production of the mammalian siderophore 2,5-DHBA involved in chelating and detoxifying iron. Based on these findings, we propose that N. gonorrhoeae can subvert the iron-limiting innate immune defenses to facilitate iron acquisition and intracellular survival.
Asunto(s)
Inmunidad Innata , Hierro/metabolismo , Macrófagos/metabolismo , Neisseria gonorrhoeae/inmunología , Regulación hacia Abajo , Humanos , Hidroxibutirato Deshidrogenasa/genética , Macrófagos/inmunología , Neisseria gonorrhoeae/metabolismo , VirulenciaRESUMEN
UNLABELLED: Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial treatments. IMPORTANCE: Gonorrhea is one of the most common bacterial sexually transmitted infections, and increasing antibiotic resistance threatens the use of currently available antimicrobial therapies. In this work, encompassing in vitro studies and in vivo studies of animal and human models of experimental genital tract infection, we document the importance of lipid A's structure, mediated by a single bacterial enzyme, LptA, in enhancing the fitness of Neisseria gonorrhoeae. The results of these studies suggest that novel agents targeting LptA may offer urgently needed prevention or treatment strategies for gonorrhea.