Filamentous fungus-produced human monoclonal antibody provides protection against SARS-CoV-2 in hamster and non-human primate models.

Monoclonal antibodies are an increasingly important tool for prophylaxis and treatment of acute virus infections like SARS-CoV-2 infection. However, their use is often restricted due to the time required for development, variable yields and high production costs, as well as the need for adaptation to newly emerging virus variants. Here we use the genetically modified filamentous fungus expression system Thermothelomyces heterothallica (C1), which has a naturally high biosynthesis capacity for secretory enzymes and other proteins, to produce a human monoclonal IgG1 antibody (HuMab 87G7) that neutralises the SARS-CoV-2 variants of concern (VOCs) Alpha, Beta, Gamma, Delta, and Omicron. Both the mammalian cell and C1 produced HuMab 87G7 broadly neutralise SARS-CoV-2 VOCs in vitro and also provide protection against VOC Omicron in hamsters. The C1 produced HuMab 87G7 is also able to protect against the Delta VOC in non-human primates. In summary, these findings show that the C1 expression system is a promising technology platform for the development of HuMabs in preventive and therapeutic medicine.

Fission yeast TORC1 prevents eIF2α phosphorylation in response to nitrogen and amino acids via Gcn2 kinase.

Serine 51 phosphorylation of the eukaryotic initiation factor-2α (eIF2α) is an important mechanism involved in blocking general protein synthesis in response to diverse types of stress. In fission yeast, three kinases (Hri1, Hri2 and Gcn2) can phosphorylate eIF2α at serine 51. In this study, we show that Tor2, as part of the TORC1 complex, prevents the phosphorylation of eIF2α in cells growing in the presence of nitrogen and amino acids. Inhibition of TORC1, either by rapamycin treatment, mutation of Tor2 or nitrogen deprivation, induces Gcn2-dependent phosphorylation of eIF2α.

AMPK phosphorylation by Ssp1 is required for proper sexual differentiation in fission yeast.

The AMP-activated protein kinase (AMPK) is a central regulator of cellular energy homeostasis, which, in response to a fall in intracellular ATP levels, activates energy-producing pathways and inhibits energy-consuming processes. Here, we report that fission yeast cells lacking AMPK activity are unable to advance entry into mitosis in response to nitrogen starvation and cannot undergo proper G1 arrest and cell differentiation. We also show that AMPK is important in the promotion of the nuclear localization and accumulation of the Ste11 transcription factor. As in animal cells, the fission yeast CaMKK ortholog (Ssp1) phosphorylates and activates the catalytic subunit of AMPK (Ssp2) in its activation loop (Thr189) when cells are starved for nitrogen or glucose. Interestingly, we found that the phosphorylation of Ssp2 on Thr189 is required for nuclear accumulation of AMPK. Our data demonstrate the existence of a signal transduction pathway activated by nutrient starvation that triggers Ssp2 phosphorylation and AMPK redistribution from the cytoplasm to the nucleus. This pathway is important to advance fission cells into mitosis and to establish a timely pre-Start G1 cell cycle arrest for mating.

The Vam6 and Gtr1-Gtr2 pathway activates TORC1 in response to amino acids in fission yeast.

The Rag family of GTPases has been implicated in the TORC1 activation in Drosophila and in mammalian cells in response to amino acids. We have investigated the role of the Rag GTPases Gtr1 and Gtr2 in TORC1 regulation in Schizosaccharomyces pombe. Fission yeast Gtr1 and Gtr2 are non-essential proteins that enhance cell growth in the presence of amino acids in the medium. The function of Gtr1 and Gtr2 in nutrient signaling is further supported by the observation that even in rich medium the deletion of either gene results in the promotion of mating, meiosis and sporulation, consistent with the downregulation of TORC1. We show that Gtr1 and Gtr2 colocalize with TORC1 in vacuoles, where TORC1 is presumably activated. Epistasis analyses indicated that Gtr1 and Gtr2 function downstream of Vam6 and upstream of TORC1 in response to amino acid signals. Our data demonstrate the existence of an evolutionarily conserved pathway with the Vam6 and Gtr1-Gtr2 pathway activating TORC1, which in turns stimulates cell growth and inhibits sexual differentiation.

TOR and PKA pathways synergize at the level of the Ste11 transcription factor to prevent mating and meiosis in fission yeast.

BACKGROUND: In the fission yeast Schizosaccharomyces pombe, the TOR (target of rapamycin) and PKA (protein kinase A) signaling transduction pathways regulate the expression of genes required for cell growth and sexual differentiation in response to the nutritional environment. Inhibition of Tor2 signaling results in the induction of genes involved in sexual differentiation, and the cells undergo mating and meiosis, even under good nutritional conditions. The same phenotype is observed in mutants in which the PKA pathway is inactive. By contrast, Tor2 overexpression or mutations that hyperactivate PKA signaling impair sexual differentiation, even under poor nutritional conditions. Accordingly, a very important question is to understand the molecular mechanism by which these two pathways coordinately regulate gene expression in response to nutrients. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that TOR and PKA pathways operate coordinately to negatively regulate sexual differentiation by inhibiting the nuclear accumulation of the Ste11 transcription factor. However, the Tor2 pathway is unable to block the nuclear localization of Ste11 under good nutritional conditions when the PKA pathway is inactive. Using microarray analyses, we found that both pathways inhibit sexual differentiation by blocking ste11-dependent gene expression. CONCLUSIONS/SIGNIFICANCE: We conclude that both the PKA and the TOR pathways inhibit Ste11 nuclear accumulation to repress Ste11-dependent gene expression. However, the PKA pathway plays a quantitatively more important role than the TOR pathway in this process.

Characterization of HMW-PBPs from the rod-shaped actinomycete Corynebacterium glutamicum: peptidoglycan synthesis in cells lacking actin-like cytoskeletal structures.

Analysis of the complete genome sequence of Corynebacterium glutamicum indicated that, in addition to ftsI, there are eight proteins with sequence motifs that are strongly conserved in penicillin binding proteins (PBPs): four genes that code for high-molecular-weight (HMW)-PBPs (PBP1a, PBP1b, PBP2a and PBP2b), two genes encoding low-molecular-weight PBPs (PBP4 and PBP4b) and two probable beta-lactamases (PBP5 and PBP6). Here, the function of the four HMW-PBPs in C. glutamicum was investigated using a combination of genetic knockouts, enhanced green fluorescent protein 2 (EGFP2) fusions and penicillin staining of membrane preparations. The four HMW-PBPs were expressed in a growing culture of C. glutamicum, but none of four pbp genes was individually essential for the growth of the bacterium, and only the simultaneous disruption of both pbp1b and pbp2b was lethal. The fused EGFP2-PBP proteins were functional in vivo, which allowed correct determination of their cellular localization. EGFP2 fusions to PBP1a, PBP1b and PBP2b localized at the poles and at the septum, whereas EGFP2-PBP2a was predominantly found at the septum. Cefsulodin treatment specifically delocalized PBP1a and PBP1b (class A HMW-PBPs), whereas mecillinam caused the specific delocalization of PBP2b and PBP2a (class B HMW-PBPs). The results provide new insight into the mechanisms involved in the synthesis of the cell wall in this bacterial species, which lacks a known actin-like cytoskeletal structure.

Morphological changes and proteome response of Corynebacterium glutamicum to a partial depletion of FtsI.

In Corynebacterium glutamicum, as in many Gram-positive bacteria, the cell division gene ftsI is located at the beginning of the dcw cluster, which comprises cell division- and cell wall-related genes. Transcriptional analysis of the cluster revealed that ftsI is transcribed as part of a polycistronic mRNA, which includes at least mraZ, mraW, ftsL, ftsI and murE, from a promoter that is located upstream of mraZ. ftsI appears also to be expressed from a minor promoter that is located in the intergenic ftsL-ftsI region. It is an essential gene in C. glutamicum, and a reduced expression of ftsI leads to the formation of larger and filamentous cells. A translational GFP-FtsI fusion protein was found to be functional and localized to the mid-cell of a growing bacterium, providing evidence of its role in cell division in C. glutamicum. This study involving proteomic analysis (using 2D SDS-PAGE) of a C. glutamicum strain that has partially depleted levels of FtsI reveals that at least 20 different proteins were overexpressed in the organism. Eight of these overexpressed proteins, which include DivIVA, were identified by MALDI-TOF. Overexpression of DivIVA was confirmed by Western blotting using anti-DivIVA antibodies, and also by fluorescence microscopy analysis of a C. glutamicum RESF1 strain expressing a chromosomal copy of a divIVA-gfp transcriptional fusion. Overexpression of DivIVA was not observed when FtsI was inhibited by cephalexin treatment or by partial depletion of FtsZ.

Characterization and use of catabolite-repressed promoters from gluconate genes in Corynebacterium glutamicum.

The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP.

Analysis of genes involved in arsenic resistance in Corynebacterium glutamicum ATCC 13032.

Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons (ars1 and ars2) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum. The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein (arsR), an arsenite permease (arsB), and an arsenate reductase (arsC); operon ars1 contains an additional arsenate reductase gene (arsC1') located immediately downstream from arsC1. Additional arsenite permease and arsenate reductase genes (arsB3 and arsC4) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1, arsB1, arsC1, arsC1', arsR2, arsB2, and arsC2 being inducible by arsenite.

Recycling of the anhydro-N-acetylmuramic acid derived from cell wall murein involves a two-step conversion to N-acetylglucosamine-phosphate.

Escherichia coli breaks down over 60% of the murein of its side wall and reuses the component amino acids to synthesize about 25% of the cell wall for the next generation. The amino sugars of the murein are also efficiently recycled. Here we show that the 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) is returned to the biosynthetic pathway by conversion to N-acetylglucosamine-phosphate (GlcNAc-P). The sugar is first phosphorylated by anhydro-N-acetylmuramic acid kinase (AnmK), yielding MurNAc-P, and this is followed by action of an etherase which cleaves the bond between D-lactic acid and the N-acetylglucosamine moiety of MurNAc-P, yielding GlcNAc-P. The kinase gene has been identified by a reverse genetics method. The enzyme was overexpressed, purified, and characterized. The cell extract of an anmK deletion mutant totally lacked activity on anhMurNAc. Surprisingly, in the anmK mutant, anhMurNAc did not accumulate in the cytoplasm but instead was found in the medium, indicating that there was rapid efflux of free anhMurNAc.

Involvement of DivIVA in the morphology of the rod-shaped actinomycete Brevibacterium lactofermentum.

In Brevibacterium lactofermentum, as in many Gram-positive bacteria, a divIVA gene is located downstream from the dcw cluster of cell-division- and cell-wall-related genes. This gene (divIVA(BL)) is mostly expressed during exponential growth, and the protein encoded, DivIVA(BL,) bears some sequence similarity to antigen 84 (Ag84) from mycobacteria and was detected with monoclonal antibodies against Ag84. Disruption experiments using an internal fragment of the divIVA(BL) gene or a disrupted divIVA(BL) cloned in a suicide conjugative plasmid were unsuccessful, suggesting that the divIVA(BL) gene is needed for cell viability in BREV: lactofermentum. Transformation of BREV: lactofermentum with a multicopy plasmid containing divIVA(BL) drastically altered the morphology of the corynebacterial cells, which became larger and bulkier, and a GFP fusion to DivIVA(BL) mainly localized to the ends of corynebacterial cells. This localization pattern, together with the overproduction phenotype, suggests that DivIVA may be important in regulating the apical growth of daughter cells.