Impact of COVID-19 vaccination on transmission risk of breakthrough infections: Lessons from adapted N95 mask sampling for emerging variants and interventions.

This study used an adapted N95 mask sampling to understand the effect of COVID-19 vaccination in the context of circulating variants on infected individuals to emit the virus into the air, a key risk factor of transmission. Mask, swab, and blood samples were collected from 92 COVID-19 patients vaccinated (Covishield/COVAXIN-partial/fully) or unvaccinated between July and September 2021 during the Delta-dominated period in Mumbai. Mask/swab samples were analyzed by reverse transcription polymerase chain reaction for viral RNA. Blood was evaluated for SARS-CoV-2 anti-spike and nucleocapsid antibody responses. At <48 h of diagnosis, 93% of the patients emitted detectable viral RNA, with 40% emitting >1000 copies in 30 min (high emitters). About 8% continued to be high emitters even after 8 days of symptom onset. No significant difference was observed in emission patterns between partial, full, and unvaccinated patients. However, when vaccinated patients were stratified based on spike protein neutralization and nucleocapsid immunoglobulin G (IgG), the group with moderate/high neutralization showed a significantly lower proportion of high emitters and viral RNA copies than the group with no/low neutralization, which further reduced in the group having antinucleocapsid IgG. In conclusion, mask sampling showed that Delta infections were associated with greater virus emission in patients, which was significantly reduced only in vaccinated patients with moderate/high SARS-CoV-2 neutralization, especially with evidence of past infection. The study demonstrated that mask sampling could be useful for understanding the transmission risk of emerging variants, screening vaccine/booster candidates, and guiding control interventions.

Genomics of postvaccination SARS-CoV-2 infections during the Delta dominated second wave of COVID-19 pandemic, from Mumbai Metropolitan Region (MMR), India.

The present study was initiated to understand the proportion of predominant variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in postvaccination infections during the Delta dominated second wave of coronavirus disease 2019 (COVID-19) in the Mumbai Metropolitan Region (MMR) in India and to understand any mutations selected in the postvaccination infections or showing association with any patient demographics. Samples were collected (n = 166) from severe/moderate/mild COVID-19 patients who were either vaccinated (COVISHIELD/COVAXIN-partial/fully vaccinated) or unvaccinated, from a city hospital and from home isolation patients in MMR. A total of 150 viral genomes were sequenced by Oxford Nanopore sequencing and the data of 136 viral genomes were analyzed for clade/lineage and for identifying mutations. The sequences belonged to three clades (21A, 21I, and 21J) and their lineage was identified as either Delta (B.1.617.2) or Delta+ (B.1.617.2 + K417N) or sub-lineages of Delta variant (AY.120/AY.38/AY.99). A total of 620 mutations were identified of which 10 mutations showed an increase in trend with time (May-October 2021). Associations of six mutations (two in spike, three in orf1a, and one in nucleocapsid) were shown with milder forms of the disease and one mutation (in orf1a) with partial vaccination status. The results indicate a trend toward reduction in disease severity as the wave progressed.

Early phase of effective treatment induces distinct transcriptional changes in Mycobacterium tuberculosis expelled by pulmonary tuberculosis patients.

Effective treatment reduces a tuberculosis patient's ability to infect others even before they test negative in sputum or culture. Currently, the basis of reduced infectiousness of the Mycobacterium tuberculosis (Mtb) with effective treatment is unclear. We evaluated changes in aerosolized bacteria expelled by patients through a transcriptomic approach before and after treatment initiation (up to 14 days) by RNA sequencing. A distinct change in the overall transcriptional profile was seen post-treatment initiation compared to pretreatment, only when patients received effective treatment. This also led to the downregulation of genes associated with cellular activities, cell wall assembly, virulence factors indicating loss of pathogenicity, and a diminished ability to infect and survive in new host cells. Based on this, we identified genes whose expression levels changed with effective treatment. The observations of the study open up avenues for further evaluating the changes in bacterial gene expression during the early phase of treatment as biomarkers for monitoring response to tuberculosis treatment regimens and provide means of identifying better correlates of Mtb transmission.

Genome Sequences of Five SARS-CoV-2 Variants from Mumbai, India, Obtained by Nanopore Sequencing.

We report here the genome sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants from five coronavirus disease 2019 (COVID-19) patients in Mumbai, India. Viral genomic RNA was isolated from nasopharyngeal swabs and/or respiratory particles from the masks of the patients. Genomic variant analysis determined 8 to 22 mutations, and the variants belong to lineages previously associated with Indian variants.

Non-invasive adapted N-95 mask sampling captures variation in viral particles expelled by COVID-19 patients: Implications in understanding SARS-CoV2 transmission.

Infectious respiratory particles expelled by SARS-CoV-2 positive patients are attributed to be the key driver of COVID-19 transmission. Understanding how and by whom the virus is transmitted can help implement better disease control strategies. Here we have described the use of a noninvasive mask sampling method to detect and quantify SARS-CoV-2 RNA in respiratory particles expelled by COVID-19 patients and discussed its relationship to transmission risk. Respiratory particles of 31 symptomatic SARS-CoV-2 positive patients and 31 asymptomatic healthy volunteers were captured on N-95 masks layered with a gelatin membrane in a 30-minute process that involved talking/reading, coughing, and tidal breathing. SARS-CoV-2 viral RNA was detected and quantified using rRT-PCR in the mask and in concomitantly collected nasopharyngeal swab (NPS) samples. The data were analyzed with respect to patient demographics and clinical presentation. Thirteen of 31(41.9%) patients showed SARS-COV-2 positivity in both the mask and NPS samples, while 16 patients were mask negative but NPS positive. Two patients were both mask and NPS negative. All healthy volunteers except one were mask and NPS negative. The mask positive patients had significantly lower NPS Ct value (26) compared to mask negative patients (30.5) and were more likely to be rapid antigen test positive. The mask positive patients could be further grouped into low emitters (expelling <100 viral copies) and high emitters (expelling >1000 viral copies). The study presents evidence for variation in emission of SARS-CoV-2 virus particles by COVID-19 patients reflecting differences in infectivity and transmission risk among individuals. The results conform to reported secondary infection rates and transmission and also suggest that mask sampling could be explored as an effective tool to assess individual transmission risks, at different time points and during different activities.

Detection of Mycobacterium tuberculosis RNA in bioaerosols from pulmonary tuberculosis patients.

BACKGROUND: Bioaerosols from pulmonary tuberculosis (PTB) patients are a quantitative predictor of transmission. Current methods involve sophisticated instruments and time-consuming techniques to assess viable TB bacteria in bioaerosols. We tested the feasibility of detecting Mycobacterium tuberculosis (Mtb) specific RNA from bioaerosols retained on TB patients' masks. METHODS: Adult PTB patients (n=33) were recruited at diagnosis before GeneXpert confirmation between April-2017 to February-2019 from private TB clinics in Mumbai. Face mask worn for 1 or 3h or N95 mask containing a cellulose acetate membrane worn for 5min by the patients were tested for the presence of Mtb RNA by quantitative PCR and bacterial load was estimated. RESULTS: Quantitative PCR targeting rpoB, sigA,16S and fgd1 and sequencing of rpoB confirmed the presence of Mtb specific RNA in mask samples including masks of two patients with unproductive sputum. Membrane samples had seven-fold higher RNA and bacterial load that correlated to bacterial load estimated by sputum GeneXpert. CONCLUSION: The study demonstrates that patient masks can be used to sample bioaerosols for detection of viable Mtb. The findings have translational value in the diagnosis of TB and monitoring Mtb variations between and within patients useful for assessing infectiousness and treatment response.

Deregulation of Genes Associated with Alternate Drug Resistance Mechanisms in Mycobacterium tuberculosis.

Alternate mechanisms of drug resistance involving intrinsic defense pathways play an important role in development of drug resistance. Deregulation of drug efflux, cellular metabolism, and DNA repair have been indicated to have effect on drug tolerance and persistence. Here we chose eight genes from these pathways to investigate their association with development of multidrug resistance (MDR). We generated mono drug resistant and MDR strains of rifampicin and isoniazid and examined the differential expression of genes belonging to efflux, DNA repair and cell wall lipid synthesis pathways. Rv1687c, recB, ppsD and embC genes showed significant (P <0.05) upregulation in mono-resistant (both rifampicin and isoniazid) as well as MDR strains. mmr showed significant upregulation with rifampicin resistance while Rv1457c showed significant upregulation only with mono-resistant strains. Highest expression change was observed with Rv1687c and ppsD. The study identified potential key genes that are significantly associated with development of drug resistance in vitro. These genes may help identify clinical strains predisposed to acquiring drug resistance in patients during the course of treatment or help in management of MDR forms of tuberculosis.

Endogenous, very small embryonic-like stem cells: critical review, therapeutic potential and a look ahead.

BACKGROUND: Both pluripotent very small embryonic-like stem cells (VSELs) and induced pluripotent stem (iPS) cells were reported in 2006. In 2012, a Nobel Prize was awarded for iPS technology whereas even today the very existence of VSELs is not well accepted. The underlying reason is that VSELs exist in low numbers, remain dormant under homeostatic conditions, are very small in size and do not pellet down at 250-280g. The VSELs maintain life-long tissue homeostasis, serve as a backup pool for adult stem cells and are mobilized under stress conditions. An imbalance in VSELs function (uncontrolled proliferation) may result in cancer. SEARCH METHODS: The electronic database 'Medline/Pubmed' was systematically searched with the subject heading term 'very small embryonic-like stem cells'. OBJECTIVE AND RATIONALE: The most primitive stem cells that undergo asymmetric cell divisions to self-renew and give rise to progenitors still remain elusive in the hematopoietic system and testes, while the presence of stem cells in ovary is still being debated. We propose to review the available literature on VSELs, the methods of their isolation and characterization, their ontogeny, how they compare with embryonic stem (ES) cells, primordial germ cells (PGCs) and iPS cells, and their role in maintaining tissue homeostasis. The review includes a look ahead on how VSELs will result in paradigm shifts in basic reproductive biology. OUTCOMES: Adult tissue-specific stem cells including hematopoietic, spermatogonial, ovarian and mesenchymal stem cells have good proliferation potential and are indeed committed progenitors (with cytoplasmic OCT-4), which arise by asymmetric cell divisions of pluripotent VSELs (with nuclear OCT-4). VSELs are the most primitive stem cells and postulated to be an overlapping population with the PGCs. Rather than migrating only to the gonads, PGCs migrate and survive in various adult body organs throughout life as VSELs. VSELs express both pluripotent and PGC-specific markers and are epigenetically and developmentally more mature compared with ES cells obtained from the inner cell mass of a blastocyst-stage embryo. As a result, VSELs readily differentiate into three embryonic germ layers and spontaneously give rise to both sperm and oocytes in vitro. Like PGCs, VSELs do not divide readily in culture, nor produce teratoma or integrate in the developing embryo. But this property of being relatively quiescent allows endogenous VSELs to survive various kinds of toxic insults. VSELs that survive oncotherapy can be targeted to induce endogenous regeneration of non-functional gonads. Transplanting healthy niche (mesenchymal) cells have resulted in improved gonadal function and live births. WIDER IMPLICATIONS: Being quiescent, VSELs possibly do not accumulate genomic (nuclear or mitochondrial) mutations and thus may be ideal endogenous, pluripotent stem cell candidates for regenerative and reproductive medicine. The presence of VSELs in adult gonads and the fact that they survive oncotherapy may obviate the need to bank gonadal tissue for fertility preservation prior to oncotherapy. VSELs and their ability to undergo spermatogenesis/neo-oogenesis in the presence of a healthy niche will help identify newer strategies toward fertility restoration in cancer survivors, delaying menopause and also enabling aged mothers to have better quality eggs.

Underlying Mechanisms that Restore Spermatogenesis on Transplanting Healthy Niche Cells in Busulphan Treated Mouse Testis.

Very small embryonic-like stem cells (VSELs) exist among spermatogonial stem cells and survive chemotherapy in both mice and human testes because of their relatively quiescent nature. Our earlier study revealed that inter-tubular transplantation of niche (Sertoli or bone marrow derived mesenchymal) cells can restore spermatogenesis from endogenous surviving VSELs. Present study was undertaken to delineate the effect of busulphan on testicular stem/germ/Sertoli cells and to comprehend the underlying mechanisms of how transplanted niche cells restore spermatogenesis. Ploidy analysis showed an increase in diploid cells on D7 and VSELs (2-6 µm; LIN-/CD45-/SCA-1+) were detected at all time-points studied and were maximum on D15 after busulphan treatment. They were visualized in cell smears, expressed nuclear NANOG and SOX2 and BrdU uptake on D15 suggested they were proliferating in response to stress induced by busulphan. Verapamil-sensitive side population detected comprised SCA-1 positive stem cells (5 ± 0.02 % in normal and 8.6 ± 2.02 % in chemoablated testis). Adverse effects of busulphan on Sertoli cells by transcriptome analysis included altered expression of Gdnf, Scf, Fgf, Bmp4, androgen binding protein, components of blood-testis-barrier and also stem cells related signaling pathways including Wnt. GFP positive transplanted cells aligned themselves as 'neo-tubules' and were visualized adjacent to 'native' germ cells depleted tubules. 'Neo-tubules' provide paracrine support to endogenous VSELs to undergo spermatogenesis. Quantitative analysis was done to track proliferation (PCNA) and differentiation (MVH) of stem cells by immuno-localization studies at different time intervals. Results provide an alternative strategy to restore spermatogenesis in cancer survivors from endogenous stem cells which needs to be further researched.

Novel Action of FSH on Stem Cells in Adult Mammalian Ovary Induces Postnatal Oogenesis and Primordial Follicle Assembly.

Adult mammalian ovary has been under the scanner for more than a decade now since it was proposed to harbor stem cells that undergo postnatal oogenesis during reproductive period like spermatogenesis in testis. Stem cells are located in the ovary surface epithelium and exist in adult and menopausal ovary as well as in ovary with premature failure. Stem cells comprise two distinct populations including spherical, very small embryonic-like stem cells (VSELs which express nuclear OCT-4 and other pluripotent and primordial germ cells specific markers) and slightly bigger ovarian germ stem cells (OGSCs with cytoplasmic OCT-4 which are equivalent to spermatogonial stem cells in the testes). These stem cells have the ability to spontaneously differentiate into oocyte-like structures in vitro and on exposure to a younger healthy niche. Bone marrow may be an alternative source of these stem cells. The stem cells express FSHR and respond to FSH by undergoing self-renewal, clonal expansion, and initiating neo-oogenesis and primordial follicle assembly. VSELs are relatively quiescent and were recently reported to survive chemotherapy and initiate oogenesis in mice when exposed to FSH. This emerging understanding and further research in the field will help evolving novel strategies to manage ovarian pathologies and also towards oncofertility.

Mouse Ovarian Very Small Embryonic-Like Stem Cells Resist Chemotherapy and Retain Ability to Initiate Oocyte-Specific Differentiation.

This study was undertaken to investigate stem cells in adult mouse ovary, the effect of chemotherapy on them and their potential to differentiate into germ cells. Very small embryonic-like stem cells (VSELs) that were SCA-1+/Lin-/CD45-, positive for nuclear octamer-binding transforming factor 4 (OCT-4), Nanog, and cell surface stage-specific embryonic antigen 1, were identified in adult mouse ovary. Chemotherapy resulted in complete loss of follicular reserve and cytoplasmic OCT-4 positive progenitors (ovarian germ stem cells) but VSELs survived. In ovarian surface epithelial (OSE) cell cultures from chemoablated ovary, proliferating germ cell clusters and mouse vasa homolog/growth differentiation factor 9-positive oocyte-like structure were observed by day 6, probably arising as a result of differentiation of the surviving VSELs. Follicle-stimulating hormone (FSH) exerted a direct stimulatory action on the OSE and induced stem cells proliferation and differentiation into premeiotic germ cell clusters during intact chemoablated ovaries culture. The FSH analog pregnant mare serum gonadotropin treatment to chemoablated mice increased the percentage of surviving VSELs in ovary. The results of this study provide evidence for the presence of potential VSELs in mouse ovaries and show that they survive chemotherapy, are modulated by FSH, and retain the ability to undergo oocyte-specific differentiation. These results show relevance to women who undergo premature ovarian failure because of oncotherapy.

Isolation and characterization of stem cells in the adult mammalian ovary.

Female mammals are born with a fixed pool of germ cells, which does not replenish during adult life. However, this has been recently challenged and adult ovaries produce oocytes throughout adult life just like sperm in the testes. Evidence is accumulating on the presence of ovarian stem cells, but the need for robust protocols to isolate, identify, further characterize, and subject them to various functionality tests is essential. Knowledge about the function and potential of ovarian stem cells is well demonstrated by various groups, but their true identity remains elusive because of the variability in the approaches used to identify them by different groups. In order to address this we have made attempts to compile our protocols to isolate, identify, characterize, and culture the stem cells using different animal models including human. Two distinct populations of stem cells exist in adult mammalian ovary, including very small embryonic-like stem cells (VSELs) and the progenitors termed ovarian germ stem cells (OGSCs). VSELs are relatively quiescent and undergo asymmetric cell division to give rise to OGSCs, which divide rapidly, occasionally form germ cell nests and undergo meiosis and differentiation into oocytes, which are surrounded by granulosa cells to assemble as primordial follicles.

Quiescent very small embryonic-like stem cells resist oncotherapy and can restore spermatogenesis in germ cell depleted mammalian testis.

Adult mouse and human testes harbor relatively quiescent, pluripotent very small embryonic-like stem cells (VSELs), in addition to actively dividing spermatogonial stem cells (SSCs). Here we report that various oncotherapy regimens in human cancer patients (n=7) and busulphan treatment (25mg/Kg body weight) in eight weeks old male mice (n=15) selectively affects actively dividing SSCs, spermatogonia, haploid germ cells and somatic microenvironment resulting in germ cell aplasia, whereas VSELs are unaffected and persist in otherwise germ cell depleted testis. Testicular VSELs are 2-5 µm in size, have high nucleo-cytoplasmic ratio, SCA-1+/CD45-/LIN- (mice), CD133+/CD45-/LIN- (human survivors of childhood cancer) and express various pluripotent transcripts including OCT-4A. SCA-1 sorted cells from busulphan treated mice testes in vitro formed small clusters suggestive of self-renewal and differentiation into progenitors, which divide rapidly. Inter-tubular random injections of syngeneic Sertoli cells (105 cells per testis, n=14) or bone marrow derived mesenchymal cells (104 cells per testis, n=16) into the germ cell depleted busulphan treated mice testes, were able to restore spermatogenesis from persisting VSELs. Transplanted Sertoli or mesenchymal cells possibly were a source of growth factors essential for VSELs differentiation. Since sperm formation occurred in situ, various epigenetic concerns associated with the 'synthetic gametes' may be eliminated in our approach. Ability of mesenchymal cells to restore spermatogenesis may benefit existing azoospermic survivors of childhood cancer who were otherwise deprived of testicular tissue cryopreservation prior to oncotherapy. Further studies are warranted to delineate the underlying mechanisms and to study quality and potential of sperm generated by this approach.

Ovarian stem cells: absence of evidence is not evidence of absence.

BACKGROUND: Lei and Spradling in a recent study published in PNAS failed to detect 'germline cysts' by elegant studies using lineage tracing approach and thus concluded that adult mouse ovaries lack stem cells. They proposed that primordial follicle pool generated during fetal life is sufficient to sustain oogenesis and that there is no renewal of oocytes during adult life. Contrary to their results, we have reported presence of very small pluripotent, embryonic-like stem cells (VSELs), their immediate descendants (OGSCs) and germ cell 'cysts' or 'nests' (formed by rapid cell division and incomplete cytokinesis) in surface epithelial cell smears of adult sheep, monkey and human ovaries. METHODS: In the present study, ovaries were collected from adult mouse (treated with 5 IU pregnant mare serum gonadotropin, PMSG) and sheep (from slaughter house) and testis from mouse treated with busulphan (25 mg/Kg). Ovarian surface epithelial (OSE) cells and testicular smears were studied for the presence of cysts. Sheep OSE smears were also used to show cytoplasmic continuity amongst the cyst cells studied by immunolocalization and confocal microscopy of stem cells specific markers OCT-4 and SSEA-4. RESULTS: Cysts were observed and confocal microscopy imaging confirmed cytoplasmic continuity amongst the cells comprising the cysts. CONCLUSIONS: Cysts represent self-renewal and clonal expansion of stem cells with incomplete cytokinesis and are a hallmark feature of stem cells. We suggest the use of PMSG stimulated mouse ovaries and use of more primitive markers like OCT-4 or STELLA rather than MVH for lineage tracing studies to conclusively show presence of stem cells by lineage-tracing studies.

Gonadotropin treatment augments postnatal oogenesis and primordial follicle assembly in adult mouse ovaries?

BACKGROUND: Follicle stimulating hormone (FSH) exerts action on both germline and somatic compartment in both ovary and testis although FSH receptors (FSHR) are localized only on the somatic cells namely granulosa cells of growing follicles and Sertoli cells in the seminiferous tubules. High levels of FSH in females are associated with poor ovarian reserve, ovarian hyper stimulation syndrome etc. and at the same time FSH acts as a survival factor during in vitro organotypic culture of ovarian cortical strips. Thus a further understanding of FSH action on the ovary is essential. We have earlier reported presence of pluripotent very small embryonic-like stem cells (VSELs express Oct-4A in addition to other pluripotent markers) and their immediate descendants 'progenitors' ovarian germ stem cells (OGSCs express Oct-4B in addition to other germ cell markers) in ovarian surface epithelium (OSE) in various mammalian species including mice, rabbit, monkey, sheep and human. Present study was undertaken to investigate the effect of pregnant mare serum gonadotropin (PMSG) on adult mice ovaries with a focus on VSELs, OGSCs, postnatal oogenesis and primordial follicle assembly. METHODS: Ovaries were collected from adult mice during different stages of estrus cycle and after 2 and 7 days of PMSG (5 IU) treatment to study histo-architecture and expression for FSHR, pluripotent stem cells , meiosis and germ cell specific markers. RESULTS: PMSG treatment resulted in increased FSHR and proliferation as indicated by increased FSHR and PCNA immunostaining in OSE and oocytes of primordial follicles (PF) besides the granulosa cells of large antral follicles. Small 1-2 regions of multilayered OSE invariably associated with a cohort of PF during estrus stage in control ovary were increased to 5-8 regions after PMSG treatment. This was associated with an increase in pluripotent transcripts (Oct-4A, Nanog), meiosis (Scp-3) and germ cells (Oct-4B, Mvh) specific markers. MVH showed positive immuno staining on germ cell nest-like clusters and at places primordial follicles appeared connected through oocytes. CONCLUSIONS: The results of the present study show that gonadotropin (PMSG) treatment to adult mouse leads to increased pluripotent stem cell activity in the ovaries, associated with increased meiosis, appearance of several cohorts of PF and their assembly in close proximity of OSE. This was found associated with the presence of germ cell nests and cytoplasmic continuity of oocytes in PF. We have earlier reported that pluripotent ovarian stem cells in the adult mammalian ovary are the VSELs which give rise to slightly differentiated OGSCs. Thus we propose that gonadotropin through its action on pluripotent VSELs augments neo-oogenesis and PF assembly in adult mouse ovaries.

Stem cell interaction with somatic niche may hold the key to fertility restoration in cancer patients.

The spontaneous return of fertility after bone marrow transplantation or heterotopic grafting of cryopreserved ovarian cortical tissue has surprised many, and a possible link with stem cells has been proposed. We have reviewed the available literature on ovarian stem cells in adult mammalian ovaries and presented a model that proposes that the ovary harbors two distinct populations of stem cells, namely, pluripotent, quiescent, very small embryonic-like stem cells (VSELs), and slightly larger "progenitor" ovarian germ stem cells (OGSCs). Besides compromising the somatic niche, oncotherapy destroys OGSCs since, like tumor cells, they are actively dividing; however VSELs persist since they are relatively quiescent. BMT or transplanted ovarian cortical tissue may help rejuvenate the ovarian niche, which possibly supports differentiation of persisting VSELs resulting in neo-oogenesis and follicular development responsible for successful pregnancies. Postnatal oogenesis in mammalian ovary from VSELs may be exploited for fertility restoration in cancer survivors including those who were earlier deprived of gametes and/or gonadal tissue cryopreservation options.

HtrA is essential for efficient secretion of recombinant proteins by Lactococcus lactis.

HtrA is a unique protease on the extracellular surface of Lactococcus lactis. It is known to take part in the proteolysis of many secreted recombinant proteins, and the mutation of htrA can lead to the complete stabilization of recombinant proteins. In this work, we have shown that htrA mutation also leads to significant reduction of the efficiency of recombinant-protein secretion. We also show that the level of HtrA can be lowered by the suppression of the acid tolerance response (ATR) in L. lactis. Instead of using an L. lactis htrA mutant, the reduction of the HtrA level in wild-type recombinant cultures of L. lactis by ATR suppression may serve as a better strategy for the production of secreted recombinant proteins.

Enhancement of recombinant streptokinase production in Lactococcus lactis by suppression of acid tolerance response.

Lactococcus lactis is a potential host for production of recombinant proteins, especially of therapeutic importance. However, in glucose-grown cultures, lowering of pH due to accumulation of lactic acid and the concomitant induction of acid tolerance response (ATR) may affect the recombinant protein produced. In this work, we have analyzed the effect of culture pH and the associated ATR on production of recombinant streptokinase. Streptokinase gene was cloned and expressed as a secretory protein in L. lactis under the control of P170 promoter. It was found to undergo degradation to form inactive products leading to low productivity. The extent of degradation and productivity of streptokinase was greatly influenced by the development of ATR, which was dependent on the pH of the culture and initial phosphate concentration of the medium. It was found that high pH and high initial phosphate concentration leads to suppression of ATR and this results in at least 2.5-fold increase in streptokinase productivity and significant decrease in degradation of streptokinase.