RESUMEN
In vitro capacitation allows for a greater understanding of the mechanisms underlying fertilization and the development of improved reproductive techniques for improving fertility rates in porcine. Tyrodes albumin lactate pyruvate (TALP) and modified Krebs Ringers Broth (m-KRB) are two medias that are commonly used in research experiments to induce capacitation in boar spermatozoa (Cañón-Beltrán et al., Theriogenology, 198, 2023 and 231; Oberlender et al., Archivos de Medicina Veterinaria, 44, 2012 and 201; Sahoo et al., International Journal of Biological Macromolecules, 241, 2023 and 124502). Moreover, understanding the morphological and functional changes in boar spermatozoa at different hours of capacitation periods might aid in the development of novel techniques for improving sperm quality and increasing the litter size. This study was carried out to investigate the effect of Tyrode albumin lactate pyruvate and modified Krebs Ringers Broth media on in vitro capacitation of HD-K75 boar spermatozoa at three different periods of incubation. A total of 24 ejaculate from four clinically healthy, 10-12 months aged HD-K75 boars, maintained at ICAR-All India Coordinated Research Project (AICRP) on pig were selected. Semen was collected by 'Simple fist' method using a portable dummy. The semen samples having 200 mL volume, 103 × 106 spermatozoa/ml concentration and 70% initial motility were selected and split into two parts and suspended in TALP and m-KRB media, respectively, and incubated for 5 h at 37°C. Seminal parameters viz. sperm viability, plasma membrane integrity and acrosomal integrity were estimated in the samples at 0, 3 and 5 h of incubation. This study revealed that there was significant variation between media in live acrosome-reacted (p < .05) and HOST-reacted (p < .01) spermatozoa, while between capacitation periods significant (p < .01) variation was observed in hyperactivated spermatozoa, live acrosome-reacted spermatozoa, HOST-reacted spermatozoa, FITC-labelled PSA, extracellular protein and sperm cholesterol. Non-significant variation was observed in total phospholipid. TALP showed overall better consequence on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. From this study, it could be concluded that both TALP and m-KRB media were virtuous to induce capacitation in HD-K75 boar spermatozoa. TALP media, however, had a better effect on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. Out of the three different periods, 3 h capacitation period resulted in significantly (p < .01) higher incidence of sperm viability, plasma membrane and acrosomal integrity in HD-K75 boar spermatozoa.
Asunto(s)
Capacitación Espermática , Espermatozoides , Animales , Masculino , Capacitación Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Porcinos , Medios de Cultivo/farmacología , Motilidad Espermática/efectos de los fármacos , Análisis de Semen/veterinariaRESUMEN
BACKGROUND: HupB is an iron-regulated protein essential for the growth of Mycobacterium tuberculosis inside macrophages. To investigate if HupB induced a dominant Th2 type immune response, we studied the effect of rHupB on PBMCs from TB patients and by infecting mouse macrophages with wild type and hupB KO mutants. METHODS: PBMCs from pulmonary TB (n = 60), extra pulmonary TB (n = 23) and healthy controls (n = 30) were stimulated with purified HupB and the cytokines secreted were assayed. The sera were screened for anti-HupB antibodies by ELISA. Mouse macrophages cell line (RAW 264.7) was infected with wild type, hupB KO and hupB-complemented strains of M. tuberculosis grown in high and low iron medium and the expression of cytokines was assayed by qRT-PCR. RESULTS: Murine macrophages infected with the hupB KO strain produced low levels of the pro-inflammatory cytokines IFN-γ, TNF-α, IL-1, and IL-18 and high levels of IL-10. HupB induced IL-6 and IL-10 production in PBMCs of TB patients and down-regulated IFN-γ and TNF-α production. The influence of HupB was remarkable in the EPTB group. CONCLUSION: HupB shifted the immune response to the Th2 type. Low IFN-γ and elevated IL-10 in EPTB patients is noteworthy.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Pulmonar , Animales , Humanos , Inmunidad , Interferón gamma , Hierro , RatonesRESUMEN
Iron-starved Mycobacterium tuberculosis utilises the carboxymycobactin-mycobactin siderophore machinery to acquire iron. These two siderophores have high affinity for ferric iron and can withdraw the metal ion from insoluble iron hydroxides and iron-binding proteins. We first reported HupB, a multi-functional mycobacterial protein to be associated with iron acquisition in M. tuberculosis. This 28 kDa cell wall protein, up regulated upon iron limitation functions as a transcriptional activator of mycobactin biosynthesis and is essential for the pathogen to survive inside macrophages. The focus of this study is to understand the role of HupB in iron uptake and transport by the carboxmycobactin-mycobactin siderophore machinery in M. tuberculosis. Experimental approaches included radiolabelled iron uptake studies by viable organisms and protein-ligand binding studies using the purified HupB and the two siderophores. Uptake of 55Fe-carboxymycobactin by wild type M. tuberculosis (WT M.tb.H37Rv) and not by the hupB KO mutant (M.tb.ΔhupB) showed that HupB is necessary for the uptake of ferri-carboxymycobactin. Additionally, the radiolabel recovery was high in HupB-incorporated liposomes upon addition of the labelled siderophore. Bioinformatic and experimental studies using spectrofluorimetry, CD analysis and surface plasmon resonance not only confirmed the binding of HupB with ferri-carboxymycobactin and ferri-mycobactin but also with free iron. In conclusion, HupB is established as a ferri- carboxymycobactin receptor and by virtue of its property to bind ferric iron, functions as a transporter of the ferric iron from the extracellular siderophore to mycobactin within the cell envelope.
Asunto(s)
Proteínas Bacterianas/metabolismo , Histonas/metabolismo , Hierro/metabolismo , Mycobacterium tuberculosis/metabolismo , Sideróforos/metabolismo , Histonas/deficienciaRESUMEN
Host cell defense against an invading pathogen depends upon various multifactorial mechanisms, several of which remain undiscovered. Here, we report a novel defense mechanism against mycobacterial infection that utilizes the histone methyltransferase, SUV39H1. Normally, a part of the host chromatin, SUV39H1, was also found to be associated with the mycobacterial bacilli during infection. Its binding to bacilli was accompanied by trimethylation of the mycobacterial histone-like protein, HupB, which in turn reduced the cell adhesion capability of the bacilli. Importantly, SUV39H1-mediated methylation of HupB reduced the mycobacterial survival inside the host cell. This was also true in mice infection experiments. In addition, the ability of mycobacteria to form biofilms, a survival strategy of the bacteria dependent upon cell-cell adhesion, was dramatically reduced in the presence of SUV39H1. Thus, this novel defense mechanism against mycobacteria represents a surrogate function of the epigenetic modulator, SUV39H1, and operates by interfering with their cell-cell adhesion ability.
Asunto(s)
Proteínas Bacterianas/inmunología , Histonas/inmunología , Macrófagos Peritoneales/inmunología , Metiltransferasas/inmunología , Mycobacterium bovis/inmunología , Proteínas Represoras/inmunología , Tuberculosis/inmunología , Animales , Humanos , Macrófagos Peritoneales/microbiología , Macrófagos Peritoneales/patología , Metilación , Ratones , Ratones Endogámicos BALB C , Células THP-1 , Tuberculosis/patología , Tuberculosis/veterinariaRESUMEN
The influence of iron levels on the transcription of the hupB gene in Mycobacterium tuberculosis is the focus of this study. Studies in our laboratory showed HupB to be co-expressed with the two siderophores in low-iron organisms. Mycobactin biosynthesis is repressed by the IdeR-Fe(2+) complex that binds the IdeR box in the mbtB promoter. Recently, we demonstrated the positive regulatory effect of HupB on mycobactin biosynthesis by demonstrating its binding to a 10 bp HupB box in the mbtB promoter. Earlier, we observed that HupB, expressed maximally in low-iron media (0.02 µg Fe ml(-1); 0.36 µM Fe) was still detectable at 8 µg Fe ml(-1) (144 µM Fe) when the siderophores were absent and complete repression was seen only at 12 µg Fe ml(-1) (216 µM Fe). In this study, we observed elevated levels of hupB transcripts in iron-limited organisms. IdeR, and not FurA, functioned as the iron regulator, by binding to two IdeR boxes in the hupB promoter. Interestingly, the 10 bp HupB box, first reported in the mbtB promoter, was identified in the hupB promoter. Using DNA footprinting and electrophoretic mobility shift assays, we demonstrated the functionality of the HupB box and the two IdeR boxes. The high hupB transcript levels expressed by the organism and the in vitro protein-DNA interaction studies led us to hypothesize the sequence of events occurring in response to changes in the intracellular iron concentration, emphasizing the roles played by IdeR and HupB in iron homeostasis.
Asunto(s)
Proteínas Bacterianas/genética , Histonas/genética , Mycobacterium tuberculosis/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Histonas/metabolismo , Hierro/metabolismo , Mycobacterium tuberculosis/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transcripción GenéticaRESUMEN
Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe(3+)-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 µg Fe ml(-1)), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.