Genome-wide CRISPR screens and their applications in infectious disease.

Inactivation or targeted disruption of a gene provides clues to assess the function of the gene in many cellular processes. Knockdown or knocking out a gene has been widely used for this purpose. However, recently CRISPR mediated genome editing has taken over the knockout/knockdown system with more precision. CRISPR technique has enabled us to perform targeted mutagenesis or genome editing to address questions in fundamental biology to biomedical research. Its application is wide in understanding the role of genes in the disease process, and response to therapy in cancer, metabolic disorders, or infectious disease. In this article, we have focused on infectious disease and how genome-wide CRISPR screens have enabled us to identify host factors involved in the process of infection. Understanding the biology of the host-pathogen interaction is of immense importance in planning host-directed therapy to improve better management of the disease. Genome-wide CRISPR screens provide strong mechanistic ways to identify the host dependency factors involved in various infections. We presented insights into genome-wide CRISPR screens conducted in the context of infectious diseases both viral and bacterial that led to better understanding of host-pathogen interactions and immune networks. We have discussed the advancement of knowledge pertaining to influenza virus, different hepatitis viruses, HIV, most recent SARS CoV2 and few more. Among bacterial diseases, we have focused on infection with life threatening Mycobacteria, Salmonella, S. aureus, etc. It appears that the CRISPR technique can be applied universally to multiple infectious disease models to unravel the role of known or novel host factors.

IL32: The multifaceted and unconventional cytokine.

Interleukin 32 is a unique intracellular cytokine which affects many cellular and physiological functions like cell death and survival, inflammation and response to pathogens. With numerous transcripts, more than one biologically active isoforms, IL32 drives its effect in diverse cellular functions. A cytokine restricted to higher mammals, it is known to fine tune multiple pathways involved in metabolic processes or infection. It modulates the immune response against diverse pathogens like Leishmania, Mycobacterium and HIV. IL32 has been associated with cancers of inflammatory nature too. It also plays an important role in chronic inflammatory diseases like RA, lung and airway disease like COPD. In this review we have discussed about identification and characterization of this non classical cytokine IL32, its structure and function at gene as well as at protein level, isoforms and their diverse functions. Role of IL32 in multiple diseases and particularly mycobacterial disease has been highlighted here. We have also summarised the genetic variants present in the IL32 gene and it's promoter region. Association of these variants, with cellular phenotype, patho-physiological conditions in different disease have also been discussed here.

Association of clade-G SARS-CoV-2 viruses and age with increased mortality rates across 57 countries and India.

Several reports have highlighted the contributions of host factors such as age, gender and co-morbidities such as diabetes, hypertension and coronary heart disease in determining COVID-19 disease severity. However, inspite of initial efforts at understanding the contributions of SARS-CoV-2 variants, most were unable to delineate causality. Hence, in this study we re-visited the contributions of different clades of viruses (G, GR and GH) along with other attributes in explaining the disparity in mortality rates among countries. A total of 26,642 high quality SARS-CoV-2 sequences were included and the A23,403G (S:D614G) variant was found to be in linkage disequilibrium with C14,408 U (RdRp: P323L). Linear regression analyses revealed increase in age [Odds ratio: 1.055 (p-value 0.000358)] and higher frequency of clade-G viruses [Odds ratio: 1.029(p-value 0.000135)] could explain 37.43% of the differences in mortality rates across the 58 countries (Multiple R-squared: 0.3743). Next, Machine-Learning algorithms LogitBoost and AdaboostM1 were applied to determine whether countries belonging to high/low mortality groups could be classified using the same attributes and accurate classification was achieved in 70.69% and 62.07% of the countries, respectively. Further, evolutionary analyses of the Indian viral population (n = 662) were carried out. Allele frequency spectrum, nucleotide diversity (π) values and negative Tajima's D values across ORFs were indicative of population expansion. Network analysis revealed the presence of two major clusters of viral haplotypes, namely, clade-G and a variant of clade L [Lv] having the RdRp:A97V amino acid change. Clade-G genomes were found to be evolving more rapidly and were also found in higher proportions in three states with highest mortality rates namely, Gujarat, Madhya Pradesh and West Bengal. Thus, the findings of this study and results from in vitro studies highlighting the role of these variants in increasing transmissibility and altering response to antivirals reflect the role of viral factors in disease prognosis.

Analysis of variants in mitochondrial genome and their putative pathogenicity in tuberculosis patients from Mizoram, North east India.

Tuberculosis caused by Mycobacterium tuberculosis is one of the main global health concerns. In this study, the entire mitochondrial genome from blood samples of tuberculosis patients was analyzed to understand the possible mtDNA variants. The potential impact of non-synonymous substitutions on protein functions were determined using prediction tools. 28 non- synonymous variants were found of which 2 variants (MT-ND2 g. A > G4824 p.T119A and MT-ND6 g. T > C14180 p.Y165C) were found to be deleterious among the cases only. Majority of the variants lie in the D-loop of the non-protein coding region of the mitochondrial DNA. We propose that mutations in the mitochondrial genome need to be validated further to understand their association with tuberculosis.

An exome wide association study of pulmonary tuberculosis patients and their asymptomatic household contacts.

Tuberculosis is a leading cause of death in India. To identify genetic variants associated with susceptibility or resistance to Mycobacterium tuberculosis infection, we have performed an exome-wide association study with 0.2 million exonic variants among 119 pairs of tuberculosis patients and their clinically asymptomatic household contacts. The strongest association was identified for rs61104666[A], a synonymous variant (p.E292E) of exon 5 of the gene SIGLEC15 (OR = 2.4, p = 1.49 × 10-5). We also found association of non-coding variants in the 3'UTR region of a gene encoding the class II human leukocyte antigens (HLAs), HLA-DRA. rs13209234[A] (minor allele frequency (MAF) = 13.8%) (OR = 0.35, P = 2.5 × 10-4) and rs3177928[A] (minor allele frequency (MAF) = 13.7%) (OR = 0.35, P = 3.3 × 10-4) were associated with protection from tuberculosis. These two SNPs, rs13209234 and rs3177928, are in complete linkage disequilibrium. These associations remained valid when additional data on freshly recruited individuals were jointly analyzed on 250 patient-control pairs. The identified gene, HLA-DRA, suggest involvement of immune regulation, indicating pathways associated with antigen presentation in tuberculosis infection.

Sputum Proteomics Reveals a Shift in Vitamin D-binding Protein and Antimicrobial Protein Axis in Tuberculosis Patients.

Existing understanding of molecular composition of sputum and its role in tuberculosis patients is variously limited to its diagnostic potential. We sought to identify infection induced sputum proteome alteration in active/non tuberculosis patients (A/NTB) and their role in altered lung patho-physiology. Out of the study population (n = 118), sputum proteins isolated from discovery set samples (n = 20) was used for an 8-plex isobaric tag for relative and absolute concentration analysis. A minimum set of protein with at least log2(ATB/NTB) >±1.0 in ATB was selected as biosignature and validated in 32 samples. Predictive accuracy was calculated from area under the receiver operating characteristic curve (AUC of ROC) using a confirmatory set (n = 50) by Western blot analysis. Mass spectrometry analysis identified a set of 192 sputum proteins, out of which a signature of ß-integrin, vitamin D binding protein:DBP, uteroglobin, profilin and cathelicidin antimicrobial peptide was sufficient to differentiate ATB from NTB. AUC of ROC of the biosignature was calculated to 0.75. A shift in DBP-antimicrobial peptide (AMP) axis in the lungs of tuberculosis patients is observed. The identified sputum protein signature is a promising panel to differentiate ATB from NTB groups and suggest a deregulated DBP-AMP axis in lungs of tuberculosis patients.

CXCL10 is overexpressed in active tuberculosis patients compared to M. tuberculosis-exposed household contacts.

INTRODUCTION: Variability in clinical outcome of tuberculosis infection is dependent, among other factors, on variation in host immunological response to the infection, which is modulated, in part by genetic variations present in the host. We undertook a study to identify host factors associated with such clinical variability. STUDY DESIGN AND METHODS: A comparative study between groups of active TB patients vs. clinically normal household contacts, family members living under the same roof with the patients for a long period of time, was carried out. We screened 22 candidate cytokines and chemokines in the plasma of 119 pairs ("discovery set") of TB patients and their asymptomatic household contacts. Identified associations were validated in an independent cohort of 78 patient-household contact pairs ("validation set"). Validated associations were further cross-validated by gene expression assays using RT-PCR and in-vitro whole blood stimulation by mycobacterial antigens ESAT6 and Rv2031c, two well-characterized antigens that are expressed in active and latent phases of disease, respectively. In a concomitant SNP association study, we have sequenced the validated gene in these patients and household contacts. RESULT: CXCL10 was found to be the most significantly (p = 0.0002) elevated chemokine - discovered and validated -- in patients' plasma compared to their household contacts. We found that CXCL10 was overexpressed by 5-fold at the RNA level in patients compared to asymptomatic household contacts (p = 0.004). On stimulation of whole blood collected from normal healthy volunteers with mycobacterial antigens ESAT6 and Rv2031c, we found that production of CXCL10 by ESAT6 was significantly higher (p = 2.8 × 10-12) than Rv2031c. The production of CXCL10 was 20-fold more than IFN-γ, the most widely validated cytokine, by ESAT6 stimulation (p = 4.6 × 10-8). One of the polymorphisms in promoter of CXCL10, rs4508917 (-1447 A > G), was identified as a proteinQTL (pQTL). Reduced expression of CXCL10 was observed among individuals with GG genotype, but the reduction was statistically significant only among controls, but not among patients. Among patients, the expression level was very high compared to the controls irrespective of the genotypes at this locus. CONCLUSION: Plasma level of CXCL10 is predictive of the active phase of TB infection.

Dissecting host factors that regulate the early stages of tuberculosis infection.

Incomplete understanding of mechanisms involved in the host-pathogen interactions constrains our efforts to eliminate tuberculosis. In many individuals, resulting from immune response to mycobacterial infection organised structures called granulomas are formed. To identify host responses that may control at least the early stages of infection, we employed an in vitro granuloma model. Here, human PBMCs were infected with live Mycobacterium tuberculosis in culture, and the appearance of granuloma-like structures was monitored over the next several days. Production of cytokines and chemokines in culture supernatants was monitored at various times, and the resulting temporal profiles were examined for possible correlations with either granuloma formation, or bacterial growth. While a positive association of TNF-α and IFN-γ secretion levels with extent of granuloma formation could clearly be identified, we were, however, unable to detect any statistically significant relationship between any cytokine/chemokine and bacterial growth. Examination of specific host cellular biochemical pathways revealed that either modulation of neutral lipid homeostasis through inhibition of the Gi-protein coupled receptor GPR109A, or regulation of host metabolic pathways through addition of vitamin D, provided a more effective means of controlling infection. A subsequent genotypic analysis for a select subset of genes belonging to pathways known to be significant for TB pathology revealed associations of polymorphisms with cytokine secretions and bacterial growth independently. Collectively therefore, the present study supports that key metabolic pathways of the host cell, rather than levels of relevant cytokines/chemokines might be more critical for regulating the intracellular mycobacterial load, in the context of granuloma formation.

Roles for Transcription Factors Sp1, NF-κB, IRF3, and IRF7 in Expression of the Human IFNL4 Gene.

The expression of a biologically active human IFNλ4 depends on the presence of a frameshift deletion polymorphism within the first exon of the interferon lambda 4 (IFNL4) gene. In this report, we use the lung carcinoma-derived cell line, A549, which is genetically viable to express a functional IFNλ4, to address transcriptional requirements of the IFNL4 gene. We show that the GC-rich DNA-binding transcription factor (TF) specificity protein 1 (Sp1) is recruited to the IFNL4 promoter and has a role in induction of gene expression upon stimulation with viral RNA mimic poly(I:C). By using RNAi and overexpression strategies, we also show key roles in IFNL4 gene expression for the virus-inducible TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), IFN regulatory factor 3 (IRF3), and IRF7. Interestingly, we also observe that overexpression of IFNλ4 influences IFNL4 promoter activity, which may further be dependent on the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway. Together, our work for the first time reports on the functional characterization of the human IFNL4 promoter.

Deregulated tyrosine-phenylalanine metabolism in pulmonary tuberculosis patients.

Metabolic profiling of biofluids from tuberculosis (TB) patients would help us in understanding the disease pathophysiology and may also be useful for the development of novel diagnostics and host-directed therapy. In this pilot study we have compared the urine metabolic profiles of two groups of subjects having similar TB symptoms and categorized as active TB (ATB, n = 21) and non-TB (NTB, n = 21) based on GeneXpert test results. Silylation, gas chromatography mass spectrometry, and standard chemometric methods were employed to identify the important molecules and deregulated metabolic pathways. Eleven active TB patients were followed up on longitudinally for comparative urine metabolic profiling with healthy controls (n = 11). A set of 42 features qualified to have a variable importance parameter score of > 1.5 of a partial least-squares discriminate analysis model and fold change of > 1.5 at p value < 0.05 between ATB and NTB. Using these variables, a receiver operating characteristics curve was plotted and the area under the curve was calculated to be 0.85 (95% CI: 0.72-0.96). Several of these variables that represent norepinephrine, gentisic acid, 4-hydroxybenzoic acid, hydroquinone, and 4-hydroxyhippuric acid are part of the tyrosine-phenylalanine metabolic pathway. In the longitudinal study we observed a treatment-dependent trend in the urine metabolome of follow-up samples, and subjects declared as clinically cured showed similar metabolic profile as those of asymptomatic healthy subjects. The deregulated tyrosine-phenylalanine axis reveals a potential target for diagnostics and intervention in TB.

Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy.

Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes approximately 60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non-HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

Loss of apolipoprotein E exacerbates the neonatal lethality of the Smith-Lemli-Opitz syndrome mouse.

The Smith-Lemli-Opitz syndrome (SLOS) is caused by a genetic defect in cholesterol biosynthesis; mutations in the enzyme 3ss-hydroxysterol Delta7 reductase (Dhcr7) lead to a failure of cholesterol (and desmosterol) synthesis, with an accumulation of precursor sterols, such as 7-dehydrocholesterol. Extensive genotype-phenotype analyses have indicated that there is considerable variation in the severity of the disease, much of which is not explained by defects in the Dhcr7 gene alone. Factors ranging from variations in maternal-fetal cholesterol transfer during pregnancy, to other genetic factors have been proposed to account for this variability. Variations at the APOE locus affect plasma cholesterol levels in humans and this polymorphic gene has been found to be associated with cardiovascular as well as neurological disorders. This locus has recently been implicated in accounting for some of the variations in SLOS. To address whether maternal hypercholesterolemia can affect fetal outcome, we tested the ability of maternal hypercholesterolemia to rescue the neonatal lethality in a mouse model of SLOS. Maternal hypercholesterolemia, induced by ApoE or Ldl-r deficiency not only failed to ameliorate the postnatal lethality, it increased the prenatal mortality of Dhcr7 deficient pups. Thus the murine data suggest that maternal loss of ApoE or Ldl-r function further exacerbates the neonatal lethality, suggesting they may play a role in maternal transfer of cholesterol to the embryo.

Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome.

Noonan syndrome is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart defects and skeletal anomalies. Increased RAS-mitogen-activated protein kinase (MAPK) signaling due to PTPN11 and KRAS mutations causes 50% of cases of Noonan syndrome. Here, we report that 22 of 129 individuals with Noonan syndrome without PTPN11 or KRAS mutation have missense mutations in SOS1, which encodes a RAS-specific guanine nucleotide exchange factor. SOS1 mutations cluster at codons encoding residues implicated in the maintenance of SOS1 in its autoinhibited form. In addition, ectopic expression of two Noonan syndrome-associated mutants induces enhanced RAS and ERK activation. The phenotype associated with SOS1 defects lies within the Noonan syndrome spectrum but is distinctive, with a high prevalence of ectodermal abnormalities but generally normal development and linear growth. Our findings implicate gain-of-function mutations in a RAS guanine nucleotide exchange factor in disease for the first time and define a new mechanism by which upregulation of the RAS pathway can profoundly change human development.

A detailed Hapmap of the Sitosterolemia locus spanning 69 kb; differences between Caucasians and African-Americans.

BACKGROUND: Sitosterolemia is an autosomal recessive disorder that maps to the sitosterolemia locus, STSL, on human chromosome 2p21. Two genes, ABCG5 and ABCG8, comprise the STSL and mutations in either cause sitosterolemia. ABCG5 and ABCG8 are thought to have evolved by gene duplication event and are arranged in a head-to-head configuration. We report here a detailed characterization of the STSL in Caucasian and African-American cohorts. METHODS: Caucasian and African-American DNA samples were genotypes for polymorphisms at the STSL locus and haplotype structures determined for this locus RESULTS: In the Caucasian population, 13 variant single nucleotide polymorphisms (SNPs) were identified and resulting in 24 different haplotypes, compared to 11 SNPs in African-Americans resulting in 40 haplotypes. Three polymorphisms in ABCG8 were unique to the Caucasian population (E238L, INT10-50 and G575R), whereas one variant (A259V) was unique to the African-American population. Allele frequencies of SNPs varied also between these populations. CONCLUSION: We confirmed that despite their close proximity to each other, significantly more variations are present in ABCG8 compared to ABCG5. Pairwise D' values showed wide ranges of variation, indicating some of the SNPs were in strong linkage disequilibrium (LD) and some were not. LD was more prevalent in Caucasians than in African-Americans, as would be expected. These data will be useful in analyzing the proposed role of STSL in processes ranging from responsiveness to cholesterol-lowering drugs to selective sterol absorption.

Induction of apoptosis by benzamide and its inhibition by aurin tricarboxylic acid (ATA) in Chinese hamster V79 cells.

To investigate the effect of benzamide and nicotinamide, well known inhibitors of poly(ADP-ribose) polymerase, in Chinese hamster V79 cells at the physiological condition of cell growth, we have tested the ability of the inhibitors to induce apoptosis. Apoptosis was detected by nuclear fragmentation, nucleosomal ladder formation, cytochrome-c release from the mitochondria and caspase-3 activation. Benzamide treatment alone increased nuclear fragmentation in dose (2.5-10 mM) and time (4-48 h)-dependent manner. Such treatment also increased nucleosomal ladders. However, 5 mM benzamide pre-treatment inhibited the nucleosomal ladders induced by gamma-irradiation indicating the role of poly(ADP-ribose) polymerase was different in irradiated cells and in un-irradiated cells. Release of cytochrome-c from the mitochondria and caspase-3 activity were also increased by such treatment. Treatment with 200 microM of aurin tricarboxylic acid (ATA), an inhibitor of DNases, inhibited the nucleosomal ladders induced by benzamide or gamma-irradiation without changing the cytochrome-c release or caspase-3 activation. This result showed that ATA inhibited the nucleosomal ladders possibly by inhibiting DNase(s) involved in apoptosis.

Identification of two differentially expressed mitochondrial genes in a methotrexate-resistant Chinese hamster cell strain derived from v79 cells using RNA fingerprinting by arbitrary primed polymerase chain reaction.

To identify genes that are differentially expressed in a methotrexate (MTX)-resistant cell strain designated as M5 that exhibits resistance to gamma radiation and a number of chemotherapeutic drugs compared to the parental Chinese hamster V79 cells, we used RNA fingerprinting by arbitrary primed polymerase chain reaction (RAP-PCR). By comparative analysis, we identified six differentially expressed transcripts that were cloned and sequenced. Two of these partial cDNA clones showed high homology to the mitochondrial genes NADH dehydrogenase subunit 1 and subunit 4. The steady-state mRNA level of both the NADH dehydrogenase subunits was about twofold higher in the M5 cell strain compared to V79 cells. Moreover, the expression of both the subunits decreased in gamma-irradiated Chinese hamster V79 cells. Cytochrome oxidase, another enzyme of the mitochondrial electron transport chain encoded in the mitochondrial genome, was also found to be overexpressed in M5 cells. All three genes are under the control of the same promoter. However, no amplification of DNA was observed. These data indicate that the alterations in mitochondrial gene expression may be involved in the recovery of irradiated cells, which may arise from transcriptional modulation of the mitochondria from the nucleus.

The rat STSL locus: characterization, chromosomal assignment, and genetic variations in sitosterolemic hypertensive rats.

BACKGROUND: Elevated plant sterol accumulation has been reported in the spontaneously hypertensive rat (SHR), the stroke-prone spontaneously hypertensive rat (SHRSP) and the Wistar-Kyoto (WKY) rat. Additionally, a blood pressure quantitative trait locus (QTL) has been mapped to rat chromosome 6 in a New Zealand genetically hypertensive rat strain (GH rat). ABCG5 and ABCG8 (encoding sterolin-1 and sterolin-2 respectively) have been shown to be responsible for causing sitosterolemia in humans. These genes are organized in a head-to-head configuration at the STSL locus on human chromosome 2p21. METHODS: To investigate whether mutations in Abcg5 or Abcg8 exist in SHR, SHRSP, WKY and GH rats, we initiated a systematic search for the genetic variation in coding and non-coding region of Abcg5 and Abcg8 genes in these strains. We isolated the rat cDNAs for these genes and characterized the genomic structure and tissue expression patterns, using standard molecular biology techniques and FISH for chromosomal assignments. RESULTS: Both rat Abcg5 and Abcg8 genes map to chromosome band 6q12. These genes span ~40 kb and contain 13 exons and 12 introns each, in a pattern identical to that of the STSL loci in mouse and man. Both Abcg5 and Abcg8 were expressed only in liver and intestine. Analyses of DNA from SHR, SHRSP, GH, WKY, Wistar, Wistar King A (WKA) and Brown Norway (BN) rat strains revealed a homozygous G to T substitution at nucleotide 1754, resulting in the coding change Gly583Cys in sterolin-1 only in rats that are both sitosterolemic and hypertensive (SHR, SHRSP and WKY). CONCLUSIONS: The rat STSL locus maps to chromosome 6q12. A non-synonymous mutation in Abcg5, Gly583Cys, results in sitosterolemia in rat strains that are also hypertensive (WKY, SHR and SHRSP). Those rat strains that are hypertensive, but not sitosterolemic (e.g. GH rat) do not have mutations in Abcg5 or Abcg8. This mutation allows for expression and apparent apical targeting of Abcg5 protein in the intestine. These rat strains may therefore allow us to study the pathophysiological mechanisms involved in the human disease of sitosterolemia.