Anti-GM-CSF autoantibodies predict outcome of cryptococcal meningitis in patients not infected with HIV: A cohort study.

OBJECTIVES: To explore the seroprevalence of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies in non-HIV cryptococcal meningitis (CM) and assess its predictive value for survival. METHODS: This is a retrospective study of 12 years of non-HIV CM. We detected serum anti-GM-CSF autoantibodies, and evaluated the clinical features and outcomes, together with the exploration of prognostic factors for 2-week and 1-year survival. RESULTS: A total of 584 non-HIV CM cases were included. 301 of 584 patients (51.5%) were phenotypically healthy. 264 Cryptococcus isolates were obtained from cerebrospinal fluid (CSF) culture, of which 251 were identified as C. neoformans species complex and 13 as C. gattii species complex. Thirty-seven of 455 patients (8.1%) tested positive for serum anti-GM-CSF autoantibodies. Patients with anti-GM-CSF autoantibodies were more susceptible to C. gattii species complex infection (66.7% vs. 6.3%; p < 0.001) and more likely to develop pulmonary mass lesions with a diameter >3 centimetres (42.9% vs. 6.5%; p 0.001). Of 584 patients 16 (2.7%) died within 2 weeks, 77 of 563 patients (13.7%) died at 1 year, and 93 of 486 patients (19.1%) lived with disabilities at 1 year. Univariant Cox regression analysis found that anti-GM-CSF autoantibodies were associated with lower 1-year survival (HR, 2.66; 95% CI, 1.34-5.27; p 0.005). Multivariable Cox proportional hazards modelling revealed that CSF cryptococcal antigen titres ≥1:1280 were associated with both, reduced 2-week and 1-year survival rates (HR, 5.44; 95% CI, 1.23-24.10; p 0.026 and HR, 5.09; 95% CI, 1.95-13.26; p 0.001). DISCUSSION: Presence of serum anti-GM-CSF autoantibodies is predictive of poor outcomes, regardless of host immune status and the causative Cryptococcus species complex.

Transcriptional inhibition of steroidogenic factor 1 in vivo in Oreochromis niloticus increased weight and suppressed gonad development.

Steroidogenic factor 1 (sf1) (officially designated as nuclear receptor subfamily 5 group A member 1 [NR5A1]) is an important regulator of gonad development. Previous studies on sf1 in fish have been limited to cloning and in vitro expression experiments. In this study, we used antisense RNA to down-regulate sf1 transcription and sf1 protein expression. Down-regulation of sf1 resulted in an increase in body weight and inhibition of gonadal development in both males and females with the consequent lower gonadosomatic index compared to fish in the control group. Hematoxylin-eosin staining of the gonads of fish with down-regulated sf1 revealed fewer seminiferous tubules and sperm in the testis of males. In addition, the oocytes were mainly stage II and many of them were atretic follicle. We conducted comparative transcriptome and proteome analyses between the sf1-down-regulated group and the control group. These analyses revealed multiple gene-protein pairs and pathways involved in regulating the observed changes, including 44 and 74 differently expressed genes and proteins in males and females, respectively. The results indicated that dysfunctional retinal metabolism and fatty acid metabolism could be causes of the observed weight gain and gonad abnormalities in sf1-down-regulated fish. These findings demonstrate the feasibility of using antisense RNA for gene editing in fish. This methodology allows the study gene function in species less amenable to gene editing as for example aquaculture species with long life cycles.

Optimal combination of temperature and photoperiod for sex steroid hormone secretion and egg development of Oreochromis niloticus as determined by response surface methodology.

For successful reproduction of farmed fish, it is important to understand the relationship between gonadal development and environmental factors such as temperature and photoperiod. In this study, we determined the effects of temperature (T) and photoperiod (Pp) on serum estradiol-17ß (E2) and progesterone (P) contents, gonadosomatic index (GSI), and oocyte development in female tilapia. We used a central composite experimental design and response surface methodology. The experimental ranges were 18-36 °C for T and 0-24 h for Pp. The results show that the quadratic effects of T and Pp were highly significant for serum E2 and P contents, GSI, and the ratio of stage III to stage II oocytes (P < 0.01), and that the linear effects of T and Pp were also significant for these indicators (P < 0.05). The T × Pp interaction significantly affected serum E2 content (P < 0.05). Serum E2 and P content, GSI, and the ratio of stage III to stage II oocytes increased and then decreased with increasing T or Pp. The best combination of T and Pp for egg development was 28.6 °C/14.29 h. We observed the part of ovarian tissue containing stage V oocytes that are about to be discharged. Shortening the photoperiod or lowering the water temperature delayed the development of ovarian tissue so that most oocytes remained at stage II, and there were many atretic follicles. There were significant positive correlations between female GSI and serum E2, P, and the ratio of stage III to stage II oocytes. The results of this study provide a reference for the regulation of temperature and photoperiod to control broodstock gonadal maturation and hormone-induced broodstock spawning.

Hypoxia-induced miR-92a regulates p53 signaling pathway and apoptosis by targeting calcium-sensing receptor in genetically improved farmed tilapia (Oreochromis niloticus).

miR-92a miRNAs are immune molecules that regulate apoptosis (programmed cell death) during the immune response. Apoptosis helps to maintain the dynamic balance in tissues of fish under hypoxia stress. The aim of this study was to explore the role and potential mechanisms of miR-92a in the liver of tilapia under hypoxia stress. We first confirmed that CaSR (encoding a calcium-sensing receptor) is a target gene of miR-92a in genetically improved farmed tilapia (GIFT) using luciferase reporter gene assays. In GIFT under hypoxia stress, miR-92a was up-regulated and CaSR was down-regulated in a time-dependent manner. Knocked-down CaSR expression led to inhibited expression of p53, TP53INP1, and caspase-3/8, reduced the proportion of apoptotic hepatocytes, and decreased the activity of calcium ions induced by hypoxia in hepatocytes. GIFT injected in the tail vein with an miR-92a agomir showed up-regulation of miR-92a and down-regulation of CaSR, p53, TP53INP1, and caspase-3/8 genes in the liver, resulting in lower serum aspartate aminotransferase and alanine aminotransferase activities under hypoxia stress. These findings suggest that stimulation of miR-92a interferes with hypoxia-induced apoptosis in hepatocytes of GIFT by targeting CaSR, thereby alleviating liver damage. These results provide new insights into the adaptation mechanisms of GIFT to hypoxia stress.

miR-34a Regulates the Activity of HIF-1a and P53 Signaling Pathways by Promoting GLUT1 in Genetically Improved Farmed Tilapia (GIFT, Oreochromis niloticus) Under Hypoxia Stress.

In fish under hypoxia stress, homeostasis can become imbalanced, leading to tissue and organ damage and decreased survival. Therefore, it is useful to explore the molecular and physiological regulation mechanisms that function in fish under hypoxia stress. The microRNA miR-34a is involved in fat and glycogen metabolism, and in apoptosis. In this study, we first verified that GLUT1, the gene encoding glucose transporter 1, is a potential target gene of miR-34a in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) by dual luciferase reporter assays. Then, we clarified the regulatory relationship between miR-34a and GLUT1 by qRT-PCR analyses. We analyzed the regulatory effects of knockdown or promotion of GLUT1 expression in vitro and in vivo in GIFT under hypoxia stress. The results confirm that GLUT1 is a target gene of miR-34a in GIFT. Down-regulation of miR-34a significantly promoted GLUT1 expression. Knockdown of GLUT1 reduced the glycogen content in GIFT liver cells, inhibited HIF-1a gene expression, up-regulated the expression of genes involved in P53 signaling pathways (P53 and CASPASE-3 genes), and accelerated hepatocyte apoptosis under hypoxia stress. Compared with the control group, the group injected in the tail vein with miR-34a antagomir showed up-regulated expression of GLUT1 in the liver, increased liver glycogen content at 96 h of hypoxia stress, down-regulated expression of P53 and CASPASE-3, and decreased serum aspartate aminotransferase and alanine aminotransferase enzyme activities. Our results provide information about the molecular regulation mechanism of miRNAs and their target genes in fish during the response to hypoxia stress.

Evaluation of low cryptococcal antigen titer as determined by the lateral flow assay in serum and cerebrospinal fluid among HIV-negative patients: a retrospective diagnostic accuracy study.

Cryptococcosis is one of the most common opportunistic infections in both immunocompetent and immunocompromised hosts. Although the cryptococcal antigen (CrAg) lateral flow assay (LFA) has been widely used in clinical settings due to its high sensitivity and specificity, the diagnostic value of a low CrAg LFA titers remains unclear. In this study, we performed a retrospective analysis of 149 HIV-negative patients with low CrAg LFA titers (≤1:10) in a Chinese tertiary hospital from January 2013 to December 2017, to evaluate the diagnostic value of low CrAg LFA titers in serum and cerebrospinal fluid (CSF) at different thresholds. Sensitivity and specificity of low CrAg LFA titers in patients with definitive diagnoses of cryptococcosis were 39.6% (95% CI, 29.7-50.1%) and 100% (95% CI, 69.2-100%), respectively, at a threshold of 1:10 in serum. A sensitivity of 72.9% (95% CI, 62.9-81.5%) and a decreased specificity of 70.0% (95% CI, 34.8-93.3%) were observed at a threshold of 1:5 in serum. No false-positive cases were identified in patients with low CrAg titers in CSF and all positive predictive values (PPVs) were 100%. Among the cases with low serum CrAg titers, lumbar puncture was performed in 97 patients and positive CSF CrAg titers were reported in 6 patients. In conclusion, the results of this study imply that low CrAg LFA titer, either in serum or CSF, is crucial for early diagnosis of cryptococcosis in HIV-negative patients, and lumbar puncture is recommended to be performed routinely for CSF testing when a positive low serum titer is reported. Cryptococcal meningitis should be considered seriously when the CSF CrAg titer is positive.

Clinically relevant mutations in mycobacterial LepA cause rifampicin-specific phenotypic resistance.

Although all wild-type bacterial populations exhibit antibiotic tolerance, bacterial mutants with higher or lower tolerant subpopulation sizes have been described. We recently showed that in mycobacteria, phenotypically-resistant subpopulations can grow in bulk-lethal concentrations of rifampicin, a first-line anti-tuberculous antibiotic targeting RNA polymerase. Phenotypic resistance was partly mediated by paradoxical upregulation of RNA polymerase in response to rifampicin. However, naturally occurring mutations that increase tolerance via this mechanism had not been previously described. Here, we used transposon insertional mutagenesis and deep sequencing (Tnseq) to investigate rifampicin-specific phenotypic resistance using two different in vitro models of rifampicin tolerance in Mycobacterium smegmatis. We identify multiple genetic factors that mediate susceptibility to rifampicin. Disruption of one gene, lepA, a translation-associated elongation factor, increased rifampicin tolerance in all experimental conditions. Deletion of lepA increased the subpopulation size that is able to grow in bulk-lethal rifampicin concentrations via upregulation of basal rpoB expression. Moreover, homologous mutations in lepA that are found in clinical Mycobacterium tuberculosis (Mtb) isolates phenocopy lepA deletion to varying degrees. Our study identifies multiple genetic factors associated with rifampicin tolerance in mycobacteria, and may allow correlation of genetic diversity of clinical Mtb isolates with clinically important phenotypes such as treatment regimen duration.

HspX promotes the polar localization of mycobacterial protein aggregates.

Misfolding of translated proteins occurs in all domains of life. In most cells, misfolded proteins coalesce in discrete aggregates at distinct cellular locations. In many bacteria, including mycobacteria, protein aggregates are located at the cellular pole. Yet the mechanism by which aggregates are sorted to the mycobacterial pole is not known. Here, we show that in Mycobacterium smegmatis, the small heat shock protein HspX plays a critical role in the polar localization of aggregates of a model fluorescent misfolded protein, GLR103. HspX itself has a polar localization, which is dependent on its N-terminal domain. In a strain deleted for hspX, GLR103 is less liable to aggregation and no longer localizes to the pole, and redirecting HspX to the septum radically disrupts the normal polar localization of GLR103 aggregates. To further investigate the role of HspX in native protein aggregation, we performed semi-quantitative mass-spectrometry of mycobacterial protein aggregates in wild-type, hspX-deleted and hspX-overexpressing strains. We identified a subset of proteins that appeared to be HspX-dependent for aggregate formation. Furthermore, we demonstrate that for validated native protein aggregates, sorting to the cellular pole following proteotoxic stress required HspX. In summary, we have identified the cellular function of HspX in Mycobacterium smegmatis as both a pro-aggregase and polar sortase.

Dietary vitamin E deficiency inhibits fat metabolism, antioxidant capacity, and immune regulation of inflammatory response in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) fingerlings following Streptococcus iniae infection.

Vitamin E plays an important role in maintaining normal metabolism and physiological functions in animals. The health of fish fingerlings directly affects the rate of disease incidence in adult fish, and healthy fingerlings ultimately result in better breeding outcomes for cultured fish. To date, no previous studies have focused on the effects vitamin E deficiency on tilapia at the fingerling stage. In this study, we investigated the effects of dietary vitamin E on the growth, fat metabolism, antioxidant capacity, and inflammatory response of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) fingerlings. Vitamin E at different concentrations (0, 20, 40, 80, 160, and 320 mg/kg) was added to the diet and GIFT were fed for 55 days. Then, the GIFT were intraperitoneally injected with Streptococcus iniae and tested for infection. Vitamin E deficiency decreased growth and increased the food conversion ratio of GIFT fingerlings. Vitamin E deficiency also reduced the white blood cell count, increased hematocrit and hemoglobin contents in the blood, increased serum aspartate aminotransferase and alanine aminotransferase activities, and increased liver stress (P < 0.05). Vitamin E deficiency inhibited fat metabolism, down-regulated the expression of genes encoding lipoprotein lipase and heart-type and liver-type fatty acid-binding proteins, and increased serum total protein and fat deposition. Vitamin E deficiency significantly decreased superoxide dismutase, glutathione peroxidase, and catalase activities, increased malondialdehyde content, and caused oxidative damage. Vitamin E deficiency also up-regulated the expression of genes encoding interleukin 1ß and tumor necrosis factor α in the head kidney, and stimulated a pro-inflammatory response. Overall, vitamin E deficiency inhibited growth, impaired fat metabolism, and disrupted the inflammatory response of GIFT fingerlings, whereas vitamin E supplementation in the diet reversed these negative effects. The diets with high concentrations of vitamin E (160-320 mg/kg) led to vitamin E accumulation in the fish tissues and rapid activation of the inflammatory response and antioxidant capacity in GIFT fingerlings exposed to S. iniae.

Rifampicin can induce antibiotic tolerance in mycobacteria via paradoxical changes in rpoB transcription.

Metrics commonly used to describe antibiotic efficacy rely on measurements performed on bacterial populations. However, certain cells in a bacterial population can continue to grow and divide, even at antibiotic concentrations that kill the majority of cells, in a phenomenon known as antibiotic tolerance. Here, we describe a form of semi-heritable tolerance to the key anti-mycobacterial agent rifampicin, which is known to inhibit transcription by targeting the ß subunit of the RNA polymerase (RpoB). We show that rifampicin exposure results in rpoB upregulation in a sub-population of cells, followed by growth. More specifically, rifampicin preferentially inhibits one of the two rpoB promoters (promoter I), allowing increased rpoB expression from a second promoter (promoter II), and thus triggering growth. Disruption of promoter architecture leads to differences in rifampicin susceptibility of the population, confirming the contribution of rifampicin-induced rpoB expression to tolerance.

Kasugamycin potentiates rifampicin and limits emergence of resistance in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation.

Most bacteria use an indirect pathway to generate aminoacylated glutamine and/or asparagine tRNAs. Clinical isolates of Mycobacterium tuberculosis with increased rates of error in gene translation (mistranslation) involving the indirect tRNA-aminoacylation pathway have increased tolerance to the first-line antibiotic rifampicin. Here, we identify that the aminoglycoside kasugamycin can specifically decrease mistranslation due to the indirect tRNA pathway. Kasugamycin but not the aminoglycoside streptomycin, can limit emergence of rifampicin resistance in vitro and increases mycobacterial susceptibility to rifampicin both in vitro and in a murine model of infection. Moreover, despite parenteral administration of kasugamycin being unable to achieve the in vitro minimum inhibitory concentration, kasugamycin alone was able to significantly restrict growth of Mycobacterium tuberculosis in mice. These data suggest that pharmacologically reducing mistranslation may be a novel mechanism for targeting bacterial adaptation.

Isobaric tag for relative and absolute quantitation-based comparative proteomic analysis of human pathogenic Prototheca zopfii genotype 2 and environmental genotype 1 strains.

BACKGROUND/PURPOSE: Prototheca species are ubiquitous achlorophyllic microalgae belonging to the family Chlorellaceae, which can cause a wide range of infections in humans and animals. Mainly in individuals with immunologic defects or trauma, Prototheca spp. can cause even lethal diseases. However, the exact pathogenic mechanism of Prototheca in causing disease remains largely unknown. To investigate the differences between pathogenic and nonpathogenic Prototheca spp. genotypes on proteome level, a nonpathogenic Prototheca zopfii genotype 1 strain, isolated from cow manure, and a human pathogenic P. zopfii genotype 2, isolated from human granulomatous lymphadenitis, were studied. METHODS: Differentially expressed proteins between the two genotypes were quantified by isobaric tag for relative and absolute quantitation-based quantitative proteomics, using liquid chromatography-tandem mass spectrometry. RESULTS: A total of 245 proteins were identified from the proteomic analysis after data filtering to eliminate low-scoring spectra. Among these, 35 proteins that displayed a significant (p<0.05) 1.5-fold change were considered as differentially expressed proteins. CONCLUSION: The differentially expressed proteins were associated with suppressed energy production and conversion, carbohydrate transport and metabolism, and enhanced translation in the genotype 2 strain, and are thus potentially relevant in the pathogenic mechanism of P. zopfii genotype 2, but need further investigation.

The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity.

Although regulation of translation fidelity is an essential process1-7, diverse organisms and organelles have differing requirements of translational accuracy8-15, and errors in gene translation serve an adaptive function under certain conditions16-20. Therefore, optimal levels of fidelity may vary according to context. Most bacteria utilize a two-step pathway for the specific synthesis of aminoacylated glutamine and/or asparagine tRNAs, involving the glutamine amidotransferase GatCAB21-25, but it had not been appreciated that GatCAB may play a role in modulating mistranslation rates. Here, by using a forward genetic screen, we show that the mycobacterial GatCAB enzyme complex mediates the translational fidelity of glutamine and asparagine codons. We identify mutations in gatA that cause partial loss of function in the holoenzyme, with a consequent increase in rates of mistranslation. By monitoring single-cell transcription dynamics, we demonstrate that reduced gatCAB expression leads to increased mistranslation rates, which result in enhanced rifampicin-specific phenotypic resistance. Consistent with this, strains with mutations in gatA from clinical isolates of Mycobacterium tuberculosis show increased mistranslation, with associated antibiotic tolerance, suggesting a role for mistranslation as an adaptive strategy in tuberculosis. Together, our findings demonstrate a potential role for the indirect tRNA aminoacylation pathway in regulating translational fidelity and adaptive mistranslation.

Tuberculosis: hey there, lonely guy!

In this issue of Molecular Cell,Samanovic et al. (2015) identify that the mycobacterial proteasomal substrate encoded by Rv1205, which appears to code for a homolog of the plant-like enzyme LONELY GUY, is responsible for proteasome-mediated nitric oxide resistance.

Protein mistranslation: friend or foe?

The translation of genes into functional proteins involves error. Mistranslation is a known cause of disease, but, surprisingly, recent studies suggest that certain organisms from all domains of life have evolved diverse pathways that increase their tolerance of translational error. Although the reason for these high error rates are not yet clear, evidence suggests that increased mistranslation may have a role in the generation of diversity within the proteome and other adaptive functions. Error rates are regulated, and there appears to be an optimal mistranslation rate that varies by organism and environmental condition. Advances in unbiased interrogation of error types and experiments involving wild organisms may help our understanding of the potentially adaptive roles for protein translation errors.

Cutaneous protothecosis in patient with diabetes mellitus and review of published case reports.

Protothecosis is an opportunistic infection caused by Prototheca, usually called as saprophytes, and is frequently found in natural and living surroundings with low virulence, but may cause chronic infection in immunocompromised individuals. We report a case of cutaneous protothecosis with zopfii var. portoricensis infection in a 66-year-old diabetic woman following hand surgery on middle right finger. Mycology study showed that smooth, creamy white, yeast-like colonies grown after necrotic tissue was cultured on Sabouraud dextrose agar at both 37 and 25°C. The organism was then identified as Prototheca zopfii var. portoricensis by molecular identification and also found from histopathology of the lesion. The lesion got improved with intravenous amphotericin B and itraconazole.