Impact of effluent parameters and vancomycin concentration on vancomycin resistant Escherichia coli and its host specific bacteriophage lytic activity in hospital effluent.

Vancomycin resistance in bacteria has been classified under high priority category by World Health Organization (WHO) and its presence in hospital effluent is reported to be increasing owing to excess antibiotics use. Among various strategies, bacteriophage has been recently considered as a promising biological agent for combating such antimicrobial resistant bacteria (ARB). However, the influence of effluent's properties on phage-ARB interaction in actual hospital effluent is not completely understood. The present works intends to study this influence of hospital effluent and its parameters on the interaction between vancomycin resistant E. coli (VRE) and its host specific bacteriophage. The isolated VRE was identified by 16S rRNA sequencing, matrix-assisted laser desorption/ionization-time of flight (MALDI - TOF) and whole genome sequencing. The infectivity of phage onto host bacteria was investigated using electron microscopic techniques, dynamic light scattering (DLS), spectrofluorophotometer and confirmed using double agar overlay method. The monovalency and polyvalency of isolated phage against various bacterial species were determined. The phage morphology was identical to T7 phage belonging to Podoviridae. The phage lysis was maximum at pH 7 (90.2%), 37 °C (91.6%) and vancomycin concentration of 50 µg/mL in both synthetic media (89.13%) and effluent (100%). At a maximum vancomycin concentration of 100 µg/mL, decrease in Ca, K, Mg and P (up to 19.70, 14.18, 28, and 15.82% respectively) concentration in effluent was observed due to phage infectivity when compared to control. The whole genome sequencing was performed and the bioinformatics analysis presented the role of mdfA gene encoding the efflux pump in causing vancomycin resistance in E. coli. It also depicted the presence of multiple genes responsible for mercury, cobalt, zinc and cadmium resistance in VRE. These results clearly indicate that bacteriophage mediated combating of VRE is possible in actual hospital effluent and can be used as one of the treatment methods.

Screening of MMV pandemic response and pathogen box compounds against pan-drug-resistant Klebsiella pneumoniae to identify potent inhibitory compounds.

Background: The recent emergence of pan-drug-resistant (PDR) K. pneumoniae strains hinders the success rate of treatment procedures for patients. High mortality, extended duration of hospitalization with high costs is associated with such infections. Discovery and identification of new drugs are inevitable to combat PDR clinical pathogens. We aim to identify and evaluate new compounds in vitro against a PDR clinical K. pneumoniae isolate using compounds of Pathogen Box and Pandemic Response Box from Medicines for Malaria Venture (MMV). Methods: The PDR strain was initially screened with the 601 compounds from both Boxes at 10 â€‹µM concentration. Formation of dormant cells against the drug activity was assessed using persister assay. MIC was determined for the drugs inhibiting PDR K. pneumoniae during initial screening. Results: Five compounds were identified to inhibit the test strain. MMV1580854 (94.60 â€‹%), MMV1579788 (94.65 â€‹%), MMV1578574 (eravacycline; 93.13 â€‹%), MMV1578566 (epetraborole; 95.29 â€‹%) and MMV1578564 (96.32 â€‹%) were able to exhibit a higher percentage of growth inhibition. Persisters were found to be growing in a range from 104 to 107 â€‹CFU/ml. Minimum inhibitory concentrations (MIC) of all compounds were ≥ 2 â€‹µM except for MMV1579788, which had a MIC of ≥ 5 â€‹µM. Conclusion: Five novel compounds were identified against the highly evolved pan-drug-resistant K. pneumoniae. Among the five, epetraborole andMMV1578564 were identified as highly potent based on the persister frequency and MICs. The pan-drug resistant clinical isolate used in this study was found to be acting differently from the reference or wild type strains against the test compounds in a previous study.

Occurrence of mcr genes and alterations in mgrB gene in intrinsic colistin- resistant Enterobacterales isolated from chicken meat samples.

BACKGROUND: Tolerance of gram negative pathogens toward last resort colistin is mediated by mcr genes and alterations in chromosomal mgrB via modification of lipopolysaccharide through the PmrAB and PhoPQ component system. Proteus sp., Morganella sp., Neisseria sp., Burkholderia sp. and Providencia sp. are intrinsic resistant to colistin drug. Recent reports have shown that colistin intrinsic resistant organisms harbor and act as reservoirs for mcr genes. AIM: To evaluate the presence of mcr-1 to mcr-5 genes and alterations in mgrB gene in intrinsic colistin-resistant gram negative bacteria isolated from chicken meat samples in Coimbatore district, Tamil Nadu, India. METHODS: One hundred chicken meat samples were collected during 2019-20. Samples were enriched and plated on MacConkey agar supplemented with colistin (2 µg/ml). The bacterial isolates were then identified using biochemical tests. DNA were extracted from isolates using the thermal lysis method. mcr-1 to mcr-5 and mgrB genes was detected using conventional PCR and agarose gel electrophoresis methods. RESULT AND CONCLUSION: The presence of mcr-1 to mcr-5 genes was found to be 23 % (23/100). mcr-1 and mcr-5 genes were not detected in sample isolates. 17/23 samples positive for mcr genes were also found to be carrying alterations in mgrB gene. Phenotypic characterization of these isolates revealed that these bacteria were belonging to colistin intrinsic resistant gram negative bacteria such as Proteus sp., Providencia sp., and Morganella sp. Intrinsic resistant bacteria could act as a potential reservoir and disseminate mcr genes to the colistin sensitive non-intrinsic pathogens of clinical importance in the environment.

Resistance patterns among drug-resistant tuberculosis patients and trends-over-time analysis of national surveillance data in Gabon, Central Africa.

OBJECTIVE: Routinely generated surveillance data are important for monitoring the effectiveness of MDR-TB control strategies. Incidence of rifampicin-resistant tuberculosis (RR-TB) is a key indicator for monitoring MDR-TB. METHODS: In a longitudinal nationwide retrospective study, 8 years (2014-2021) of sputum samples from presumptively drug-resistant tuberculosis patients from all regions of Gabon were referred to the national tuberculosis reference laboratory. Samples were analysed using GeneXpert MTB/RIF and Genotype MTBDRsl version 2/Line Probe Assay. RESULTS: Of 3057 sputum samples from presumptive tuberculosis patients, both from local hospital and from referral patients, 334 were RR-TB. The median patient age was 33 years (interquartile range 26-43); one third was newly diagnosed drug-resistant tuberculosis patients; one-third was HIV-positive. The proportion of men with RR-TB was significantly higher than that of women (55% vs 45%; p < 0.0001). Patients aged 25-35 years were most affected (32%; 108/334). The cumulative incidence of RR-TB was 17 (95% CI 15-19)/100,000 population over 8 years. The highest incidences were observed in 2020 and 2021. A total of 281 samples were analysed for second-line drug resistance. The proportions of study participants with MDR-TB, pre-XDR-TB and XDR-TB were 90.7% (255/281), 9% (25/281) and 0.3% (1/281), respectively. The most-common mutations in fluoroquinolones resistance isolates was gyrA double mutation gyrA MUT3B and MUT3C (23%; 4/17). Most (64%; 6/8) second-line injectable drugs resistance isolates were characterised by missing both rrs WT2 and MUT2 banding. CONCLUSION: The increasing incidence of MDR-TB infection in Gabon is alarming. It is highest in the 25-35 years age category. The incidence of MDR-TB infection in treatment-naïve patients calls for case finding and contact tracing strategy improvement.

Non-typhoidal Salmonella causing urinary tract infection in a young male with renal calculi - a case report and comprehensive review.

Introduction: Non-typhoidal Salmonella (NTS) causes urinary tract infections infrequently and are usually associated with presence of genitourinary abnormalities. Case presentation: We report a case of immunocompetent male in his early 20 s with phimosis presented with history of dysuria and burning micturition for 4 months. A renal ultrasonography showed presence of bilateral intrarenal calculi. Urine analysis revealed presence of non-typhoidal Salmonella. Automated identification systems performed poorly in identification of serotype. On serotyping, it was identified as Salmonella enteritidis in the referral centre. The patient was managed with oral antibiotics. Conclusion: This report highlights the issues of inaccurate identification of NTS even with advanced automated systems and early initiation of therapy based on the knowledge of local susceptibility patterns. UTI in immunocompetent individuals by non-typhoidal Salmonella should always be investigated further to rule out genitourinary abnormalities and appropriate antibiotics must be started to avoid chronicity and complications.

In-silico analysis of Covid-19 genome sequences of Indian origin: Impact of mutations in identification of SARS-Co-V2.

Covid-19 disease caused by SARS-CoV-2 is still being transmitted in developed and developing countries irrespective of healthcare setups. India with 1.3 billion people in the world is severely affected by Covid-19 with 11.3 million cases and 157 000 deaths so far. We have assessed the mismatches in WHO recommended rRT-PCR assays primer and probe binding regions against SARS-CoV-2 Indian genome sequences through in-silico bioinformatics analysis approach. Primers and probe sequences belonging to CN-CDC-ORF1ab from China and HKU-ORF1b from Hong Kong targeting ORF1ab gene while NIH-TH-N from Thailand, HKU-N from Hong Kong and US-CDCN-2 from USA targeting N genes displayed accurate matches (>98.3%) with the 2019 novel corona virus sequences from India. On the other hand, none of the genomic sequences displayed exact match with the primer/probe sequences belonging to Charité-ORF1b from Germany targeting ORF1ab gene. We think it will be worthwhile to release this information to the clinical and medical communities working in Indian Covid-19 frontline taskforce to tackle the recently emerging Covid-19 outbreaks as of March-2021.

Species and genotype diversity of Plasmodium in malaria patients from Gabon analysed by next generation sequencing.

BACKGROUND: Six Plasmodium species are known to naturally infect humans. Mixed species infections occur regularly but morphological discrimination by microscopy is difficult and multiplicity of infection (MOI) can only be evaluated by molecular methods. This study investigated the complexity of Plasmodium infections in patients treated for microscopically detected non-falciparum or mixed species malaria in Gabon. METHODS: Ultra-deep sequencing of nucleus (18S rRNA), mitochondrion, and apicoplast encoded genes was used to evaluate Plasmodium species diversity and MOI in 46 symptomatic Gabonese patients with microscopically diagnosed non-falciparum or mixed species malaria. RESULTS: Deep sequencing revealed a large complexity of confections in patients with uncomplicated malaria, both on species and genotype levels. Mixed infections involved up to four parasite species (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale curtisi, and P. ovale wallikeri). Multiple genotypes from each species were determined from the asexual 18S rRNA gene. 17 of 46 samples (37%) harboured multiple genotypes of at least one Plasmodium species. The number of genotypes per sample (MOI) was highest in P. malariae (n = 4), followed by P. ovale curtisi (n = 3), P. ovale wallikeri (n = 3), and P. falciparum (n = 2). The highest combined genotype complexity in samples that contained mixed-species infections was seven. CONCLUSIONS: Ultra-deep sequencing showed an unexpected breadth of Plasmodium species and within species diversity in clinical samples. MOI of P. ovale curtisi, P. ovale wallikeri and P. malariae infections were higher than anticipated and contribute significantly to the burden of malaria in Gabon.

Molecular surveillance of Plasmodium falciparum drug resistance in the Republic of Congo: four and nine years after the introduction of artemisinin-based combination therapy.

BACKGROUND: Resistance to anti-malarial drugs hinders efforts on malaria elimination and eradication. Following the global spread of chloroquine-resistant parasites, the Republic of Congo adopted artemisinin-based combination therapy (ACT) in 2006 as a first-line treatment for uncomplicated malaria. To assess the impacts after implementation of ACT, a molecular surveillance for anti-malarial drug resistance was conducted in Congo 4 and 9 years after the introduction of ACT. METHODS: Blood samples of 431 febrile children aged 1-10 years were utilized from two previous studies conducted in 2010 (N = 311) and 2015 (N = 120). All samples were screened for malaria parasites using nested PCR. Direct sequencing was used to determine the frequency distribution of genetic variants in the anti-malarial drug-resistant Plasmodium falciparum genes (Pfcrt, Pfmdr1, Pfatp6, Pfk13) in malaria-positive isolates. RESULTS: One-hundred and nineteen (N = 70 from 2010 and N = 49 from 2015) samples were positive for P. falciparum. A relative decrease in the proportion of chloroquine-resistant haplotype (CVIET) from 100% in 2005, 1 year before the introduction and implementation of ACT in 2006, to 98% in 2010 to 71% in 2015 was observed. Regarding the multidrug transporter gene, a considerable reduction in the frequency of the mutations N86Y (from 73 to 27%) and D1246Y (from 22 to 0%) was observed. However, the prevalence of the Y184F mutation remained stable (49% in 2010 compared to 54% in 2015). Isolates carrying the Pfatp6 H243Y was 25% in 2010 and this frequency was reduced to null in 2015. None of the parasites harboured the Pfk13 mutations associated with prolonged artemisinin clearance in Southeast Asia. Nevertheless, 13 new Pfk13 variants are reported among the investigated isolates. CONCLUSION: The implementation of ACT has led to the decline in prevalence of chloroquine-resistant parasites in the Republic of Congo. However, the constant prevalence of the PfMDR1 Y184F mutation, associated with lumefantrine susceptibility, indicate a selective drug pressure still exists. Taken together, this study could serve as the basis for epidemiological studies monitoring the distribution of molecular markers of artemisinin resistance in the Republic of Congo.

Natural infection of Plasmodium brasilianum in humans: Man and monkey share quartan malaria parasites in the Venezuelan Amazon.

BACKGROUND: The quartan malaria parasite Plasmodium malariae is the widest spread and best adapted human malaria parasite. The simian Plasmodium brasilianum causes quartan fever in New World monkeys and resembles P. malariae morphologically. Since the genetics of the two parasites are nearly identical, differing only in a range of mutations expected within a species, it has long been speculated that the two are the same. However, no naturally acquired infection with parasites termed as P. brasilianum has been found in humans until now. METHODS: We investigated malaria cases from remote Yanomami indigenous communities of the Venezuelan Amazon and analyzed the genes coding for the circumsporozoite protein (CSP) and the small subunit of ribosomes (18S) by species-specific PCR and capillary based-DNA sequencing. FINDINGS: Based on 18S rRNA gene sequencing, we identified 12 patients harboring malaria parasites which were 100% identical with P. brasilianum isolated from the monkey, Alouatta seniculus. Translated amino acid sequences of the CS protein gene showed identical immunodominant repeat units between quartan malaria parasites isolated from both humans and monkeys. INTERPRETATION: This study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts.

Antimalarial compounds in Phase II clinical development.

INTRODUCTION: Malaria is a major health problem in endemic countries and chemotherapy remains the most important tool in combating it. Treatment options are limited and essentially rely on a single drug class - the artemisinins. Efforts are ongoing to restrict the evolving threat of artemisinin resistance but declining sensitivity has been reported. Fueled by the ambitious aim of malaria eradication, novel antimalarial compounds, with improved properties, are now in the progressive phase of drug development. AREAS COVERED: Herein, the authors describe antimalarial compounds currently in Phase II clinical development and present the results of these investigations. EXPERT OPINION: Thanks to recent efforts, a number of promising antimalarial compounds are now in the pipeline. First safety data have been generated for all of these candidates, although their efficacy as antimalarials is still unclear for most of them. Of particular note are KAE609, KAF156 and DSM265, which are of chemical scaffolds new to malaria chemotherapy and would truly diversify antimalarial options. Apart from SAR97276, which also has a novel chemical scaffold that has had its development stopped, all other compounds in the pipeline belong to already known substance classes, which have been chemically modified. At this moment in time, there is not one standout compound that will revolutionize malaria treatment but several compounds that will add to its control in the future.

Complex expression dynamics and robustness in C. elegans insulin networks.

Gene families expand by gene duplication, and resulting paralogs diverge through mutation. Functional diversification can include neofunctionalization as well as subfunctionalization of ancestral functions. In addition, redundancy in which multiple genes fulfill overlapping functions is often maintained. Here, we use the family of 40 Caenorhabditis elegans insulins to gain insight into the balance between specificity and redundancy. The insulin/insulin-like growth factor (IIS) pathway comprises a single receptor, DAF-2. To date, no single insulin-like peptide recapitulates all DAF-2-associated phenotypes, likely due to redundancy between insulin-like genes. To provide a first-level annotation of potential patterns of redundancy, we comprehensively delineate the spatiotemporal and conditional expression of all 40 insulins in living animals. We observe extensive dynamics in expression that can explain the lack of simple patterns of pairwise redundancy. We propose a model in which gene families evolve to attain differential alliances in different tissues and in response to a range of environmental stresses.

The C-terminal PDZ-binding motif in the Kv1.5 potassium channel governs its modulation by the Na+/H+ exchanger regulatory factor 2.

Kv1.5 belongs to the family of voltage-gated potassium (Kv) channels and contains a N- and a C-terminal PDZ-binding motif that might be recognized by PDZ domains on the scaffold proteins NHERF1 and NHERF2. Expression studies in Xenopus oocytes demonstrated that NHERF1 and NHERF2 activate Kv1.5, an effect requiring the C-terminal PDZ-binding motif on Kv1.5. NHERF2 enhances Kv1.5 activity and cell surface expression as determined by electrophysiology and immunoassays. NHERF2 elevates Kv1.5 abundance at the plasma membrane by decreasing channel internalization as proven by Brefeldin A experiments. Kv1.5 is stimulated by the serum and glucocorticoid inducible kinase SGK1, a kinase known to interact with the second PDZ domain of NHERF2. This study aims to identify if SGK1 and NHERF2 synergize to increase Kv1.5 currents. Expression of NHERF2 potentiated SGK1-mediated Kv1.5 activation, which was significantly attenuated by deletion of the second PDZ domain in NHERF2. Specificity of observed effects was verified by evaluating the influence of NHERFs on Kv1.3, a known SGK1 target that contains an internal PDZ binding motif. In summary, our results suggest that NHERFs might participate in the regulation of electrical excitability in part by controlling Kv1.5 surface abundance and by clustering signal transduction molecules to the channel.

Modulation of the voltage-gated potassium channel Kv1.5 by the SGK1 protein kinase involves inhibition of channel ubiquitination.

The serum and glucocorticoid inducible kinase SGK1 is involved in dexamethasone-induced inhibition of insulin secretion by increasing voltage-gated potassium channel (Kv) activity. SGK1 upregulates the Kv1.5 channel but the precise mechanism underlying the SGK1 dependent regulation of Kv1.5 has not been defined yet. The present study explored the signal transduction processes involved. Expression studies in Xenopus oocytes revealed that SGK1 promotes channel activity by interfering with the Nedd4-2 ubiquitination pathway, irrespective of the presence of putative SGK1 phosphorylation sites on Kv1.5. Expression of the ubiquitin ligase Nedd4-2 declined Kv1.5 currents by ubiquitinating and thereby reducing Kv1.5 plasma membrane expression. Increasing concentrations of SGK1 gradually compensated the inhibiting effect of Nedd4-2 on Kv1.5. Enhanced Kv1.5 surface abundance by SGK1 reflects decreased channel internalization as indicated by Brefeldin A experiments. In conclusion, Kv1.5 upregulation by SGK1 involves inhibition of channel ubiquitination by Nedd4-2 that leads to Kv1.5 stabilization in the plasma membrane. Our results suggest that the kinase might participate in the regulation of insulin secretion in part by controlling Kv1.5 surface abundance.

The peptide transporter PEPT2 is targeted by the protein kinase SGK1 and the scaffold protein NHERF2.

PEPT1 and PEPT2 are members of the family of proton-dependent oligopeptide transporters that mediate electrogenic uphill transport of small peptides and peptidomimetics into a variety of cells. Kinetic properties and substrate recognition sites of those transporters have been well defined previously. Little is known, however, about regulation of those transporters. Both PEPT isoforms contain putative phosphorylation sites for the serum and glucocorticoid inducible kinase SGK1 and a C-terminal PDZ binding motif that might be recognized by PDZ domains of the Na(+)/H(+) exchanger regulatory factors NHERF1 and NHERF2. Thus, the present study attempted to clarify the role of SGK1 and NHERFs in the modulation of PEPT isoforms. Expression studies in Xenopus oocytes with subsequent electrophysiology and immunoassays revealed that SGK1 and NHERF2, but not the NHERF1 isoform specifically enhance PEPT2 function and surface abundance. The kinase is effective through phosphorylation of (185)Ser within the SGK1 consensus site, since disruption of this site prevented transporter modulation by the kinase. NHERF2 failed to regulate the C-terminal deletion mutant (PEPT2DeltaC) indicating that the C-terminal PDZ-binding motif in PEPT2 governs transport modulation by NHERF2. Coexpression of NHE3 stimulates PEPT2 activity to a similar extent as coexpression of NHERF2. Dynasore experiments demonstrated that SGK1 and NHERF2 activate PEPT2 by stabilizing the transporter at the cell surface. In conclusion, the present results reveal two novel PEPT2 posttranslational modulators, SGK1 and NHERF2, which might regulate transport of oligopeptides and peptidomimetic drugs.

Alterations in the cytoplasmic domain of CLCN2 result in altered gating kinetics.

Mutations in the human ClC-2 Cl(-) channel have been described to influence its function dramatically. To test for naturally occurring gene variants in a human population and their functionality, all 24 CLCN2 exons from a Central African population were sequenced. Six single amino acid exchanges in the intracellular N-terminus (P48R, R68H), in the pore domain (G199A), or in the intracellular C-terminus (R646Q, R725W, R747H) were identified at low frequency. Heterologous expression of these polymorphisms in Xenopus laevis oocytes demonstrated their functional significance as determined by two-electrode voltage-clamp. The polymorphisms R68H, R725W, and R747H exhibited faster voltage-stimulated gating as compared to the wild type channel, resulting in higher steady state currents of R725W. Probably due to decreased surface expression P48R, R68H, and R646Q mutants generated lower currents than the wild type channels. The inward currents of the mutated channels R725W, R747H, and G199A failed to increase during hypotonic swelling, a defect paralleled by impaired swelling-accelerated voltage-gating in one mutant (G199A). In conclusion, the Africans' gene pool comprises CLCN2 gene variants in the N-terminus, the C-terminus or the pore domain that affect surface expression and voltage- or cell-swelling-stimulated channel gating.

Role of SGK1 kinase in regulating glucose transport via glucose transporter GLUT4.

Insulin stimulates glucose transport into muscle and fat cells by enhancing GLUT4 abundance in the plasma membrane through activation of phosphatidylinositol 3-kinase (PI3K). Protein kinase B (PKB) and PKCzeta are known PI3K downstream targets in the regulation of GLUT4. The serum- and glucocorticoid-inducible kinase SGK1 is similarly activated by insulin and capable to regulate cell surface expression of several metabolite transporters. In this study, we evaluated the putative role of SGK1 in the modulation of GLUT4. Coexpression of the kinase along with GLUT4 in Xenopus oocytes stimulated glucose transport. The enhanced GLUT4 activity was paralleled by increased transporter abundance in the plasma membrane. Disruption of the SGK1 phosphorylation site on GLUT4 ((S274A)GLUT4) abrogated the stimulating effect of SGK1. In summary, SGK1 promotes glucose transporter membrane abundance via GLUT4 phosphorylation at Ser274. Thus, SGK1 may contribute to the insulin and GLUT4-dependent regulation of cellular glucose uptake.

SGK1 kinase upregulates GLUT1 activity and plasma membrane expression.

Phosphatidylinositol 3-kinase (PI3 kinase) inhibition disrupts the ability of insulin to stimulate GLUT1 and GLUT4 translocation into the cell membrane and thus glucose transport. The effect on GLUT4 but not on GLUT1 is mediated by activation of protein kinase B (PKB). The serum- and glucocorticoid-inducible kinase SGK1, a further kinase downstream of PI3 kinase, regulates several transporters by enhancing their plasma membrane abundance. GLUT1 contains a consensus site ((95)Ser) for phosphorylation by SGK1. Thus, the present study investigated whether GLUT1 is regulated by the kinase. Tracer-flux studies in Xenopus oocytes and HEK-293 cells demonstrated that GLUT1 transport is enhanced by constitutively active (S422D)SGK1. The effect requires the kinase catalytical activity since the inactive mutant (K127N)SGK1 failed to modulate GLUT1. GLUT1 stimulation by (S422D)SGK1 is not due to de novo protein synthesis but rather to an increase of the transporter's abundance in the plasma membrane. Kinetic analysis revealed that SGK1 enhances maximal transport rate without altering GLUT1 substrate affinity. These observations suggest that SGK1 regulates GLUT1 and may contribute to or account for the PI3 kinase-dependent but PKB-independent stimulation of GLUT1 by insulin.

The serine/threonine kinases SGK1, 3 and PKB stimulate the amino acid transporter ASCT2.

The human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2/SLC1A5) plays an important role in the transport of neutral amino acids in epithelial cells. The serine and threonine kinases SGK1-3 and protein kinase B have been implicated in the regulation of several members of the SLC1 transporter family by enhancing their plasma membrane abundance. The present study explored whether those kinases modulate hASCT2. In Xenopus oocytes heterologously expressing hASCT2, coexpression of constitutively active (S422D)SGK1, (S419D)SGK3 or (T308DS473D)PKB upregulated the transporter activity. The stimulation requires the catalytical activity of the kinases since the inactive mutants (K127N)SGK1, (K191N)SGK3, and (T308AS473A)PKB failed to modulate the transporter. According to kinetic analysis and chemiluminescence assays, SGK1 and SGK3 modulate hASCT2 by enhancing the transporter abundance in the plasma membrane. As SGK1, 3 and PKB are activated by insulin and IGF1, the described mechanisms presumably participate in the hormonal stimulation of cellular amino acid uptake.