Membrane proteome of the thermoalkaliphile Caldalkalibacillus thermarum TA2.A1.

Proteomics has greatly advanced the understanding of the cellular biochemistry of microorganisms. The thermoalkaliphile Caldalkalibacillus thermarum TA2.A1 is an organism of interest for studies into how alkaliphiles adapt to their extreme lifestyles, as it can grow from pH 7.5 to pH 11. Within most classes of microbes, the membrane-bound electron transport chain (ETC) enables a great degree of adaptability and is a key part of metabolic adaptation. Knowing what membrane proteins are generally expressed is crucial as a benchmark for further studies. Unfortunately, membrane proteins are the category of proteins hardest to detect using conventional cellular proteomics protocols. In part, this is due to the hydrophobicity of membrane proteins as well as their general lower absolute abundance, which hinders detection. Here, we performed a combination of whole cell lysate proteomics and proteomics of membrane extracts solubilised with either SDS or FOS-choline-12 at various temperatures. The combined methods led to the detection of 158 membrane proteins containing at least a single transmembrane helix (TMH). Within this data set we revealed a full oxidative phosphorylation pathway as well as an alternative NADH dehydrogenase type II (Ndh-2) and a microaerophilic cytochrome oxidase ba3. We also observed C. thermarum TA2.A1 expressing transporters for ectoine and glycine betaine, compounds that are known osmolytes that may assist in maintaining a near neutral internal pH when the external pH is highly alkaline.

Genomic analysis of Caldalkalibacillus thermarum TA2.A1 reveals aerobic alkaliphilic metabolism and evolutionary hallmarks linking alkaliphilic bacteria and plant life.

The aerobic thermoalkaliphile Caldalkalibacillus thermarum strain TA2.A1 is a member of a separate order of alkaliphilic bacteria closely related to the Bacillales order. Efforts to relate the genomic information of this evolutionary ancient organism to environmental adaptation have been thwarted by the inability to construct a complete genome. The existing draft genome is highly fragmented due to repetitive regions, and gaps between and over repetitive regions were unbridgeable. To address this, Oxford Nanopore Technology's MinION allowed us to span these repeats through long reads, with over 6000-fold coverage. This resulted in a single 3.34 Mb circular chromosome. The profile of transporters and central metabolism gives insight into why the organism prefers glutamate over sucrose as carbon source. We propose that the deamination of glutamate allows alkalization of the immediate environment, an excellent example of how an extremophile modulates environmental conditions to suit its own requirements. Curiously, plant-like hallmark electron transfer enzymes and transporters are found throughout the genome, such as a cytochrome b6c1 complex and a CO2-concentrating transporter. In addition, multiple self-splicing group II intron-encoded proteins closely aligning to those of a telomerase reverse transcriptase in Arabidopsis thaliana were revealed. Collectively, these features suggest an evolutionary relationship to plant life.

Quantification of T-Cell and B-Cell Replication History in Aging, Immunodeficiency, and Newborn Screening.

Quantification of T-cell receptor excision circles (TRECs) has impacted on human T-cell research, but interpretations on T-cell replication have been limited due to the lack of a genomic coding joint. We here overcome this limitation with multiplex TRG rearrangement quantification (detecting ~0.98 alleles per TCRαß+ T cell) and the HSB-2 cell line with a retrovirally introduced TREC construct. We uncovered <5 cell divisions in naive and >10 cell divisions in effector memory T-cell subsets. Furthermore, we show that TREC dilution with age in healthy adults results mainly from increased T cell replication history. This proliferation was significantly increased in patients with predominantly antibody deficiency. Finally, Guthrie cards of neonates with Down syndrome have fewer T and B cells than controls, with similar T-cell and slightly higher B-cell replication. Thus, combined analysis of TRG coding joints and TREC signal joints can be utilized to quantify in vivo T-cell replication, and has direct applications for research into aging, immunodeficiency, and newborn screening.

Impaired STAT3-Dependent Upregulation of IL2Rα in B Cells of a Patient With a STAT1 Gain-of-Function Mutation.

Heterozygous STAT1 gain-of-function (GOF) mutations form the most common genetic cause of chronic mucocutaneous candidiasis (CMC). In such patients, increased STAT1 function leads to impaired STAT3-dependent activation of IL-17A and IL-17F in T cells, thereby causing impaired Th17 responses to Candida. In spite of the critical role of STAT3 in IL-21 signaling in B cells, nearly all STAT1 GOF patients have normal or high serum IgG. We here present a 44 year-old male with childhood onset of CMC and antibody deficiency since early adulthood. Sequence analysis of STAT1 revealed a heterozygous missense mutation in the coiled-coil domain (p.D168E), which resulted in increased STAT1 phosphorylation of B-cells activated with IFNα and IFNγ. IL-21 induced STAT3 phosphorylation and nuclear localization were normal, but resulted in impaired upregulation of IL2Rα. This newly identified B-cell intrinsic impairment of STAT3 function could underlie the progressive development of hypogammaglobulinemia. Considering the high risk of bronchiectasis and irreversible organ damage, this case illustrates the need for monitoring of IgG levels and/or function in adult patients with STAT1 GOF mutations.