Quantitative proteomics analysis of an ethanol- and a lactate-producing mutant strain of Synechocystis sp. PCC6803

BACKGROUND: This study aimed at exploring the molecular physiological consequences of a major redirection of carbon flow in so-called cyanobacterial cell factories: quantitative whole-cell proteomics analyses were carried out on two (14)N-labelled Synechocystis mutant strains, relative to their (15)N-labelled wild-type counterpart. Each mutant strain overproduced one specific commodity product, i.e. ethanol or lactic acid, to such an extent that the majority of the incoming CO2 in the organism was directly converted into the product. RESULTS: In total, 267 proteins have been identified with a significantly up- or down-regulated expression level. In the ethanol-producing mutant, which had the highest relative direct flux of carbon-to-product (>65%), significant up-regulation of several components involved in the initial stages of CO2 fixation for cellular metabolism was detected. Also a general decrease in abundance of the protein synthesizing machinery of the cells and a specific induction of an oxidative stress response were observed in this mutant. In the lactic acid overproducing mutant, that expresses part of the heterologous l-lactate dehydrogenase from a self-replicating plasmid, specific activation of two CRISPR associated proteins, encoded on the endogenous pSYSA plasmid, was observed. RT-qPCR was used to measure, of nine of the genes identified in the proteomics studies, also the adjustment of the corresponding mRNA level. CONCLUSION: The most striking adjustments detected in the proteome of the engineered cells were dependent on the specific product formed, with, e.g. more stress caused by lactic acid- than by ethanol production. Up-regulation of the total capacity for CO2 fixation in the ethanol-producing strain was due to hierarchical- rather than metabolic regulation. Furthermore, plasmid-based expression of heterologous gene(s) may induce genetic instability. For selected, limited, number of genes a striking correlation between the respective mRNA- and the corresponding protein expression level was observed, suggesting that for the expression of these genes regulation takes place primarily at the level of gene transcription

The ESX-5 System of Pathogenic Mycobacteria Is Involved In Capsule Integrity and Virulence through Its Substrate PPE10

Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulatio

Surface labelling of capsular protein EspE and capsular glycans is dependent on ESX-5.

<p>A) Parental strains of wild-type <i>M</i>. <i>marinum</i> E11, or isogenic mutant strains <i>espG</i>_<i>5</i>::<i>tn</i>, <i>eccCb</i>_<i>1</i>::<i>tn</i> and <i>ppe10</i>::<i>tn</i> expressing pSMT3::<i>mCherry</i>, were grown in the presence or absence of Tween-80 and labeled with the α-EspE antibody and a FITC labeled secondary antibody. Subsequently, cells were analyzed by flow cytometry analysis. High levels of surface expression of EspE could be detected in the absence of Tween-80 in both wild-type <i>M</i>. <i>marinum</i> and the <i>espG</i>_<i>5</i>::<i>tn</i> mutant strain, while <i>ppe10</i>::<i>tn</i> showed an intermediate EspE surface labelling. When grown in the presence of Tween-80, surface labeling of EspE was almost completely lost and reduced to similar levels of the ESX-1 mutant strain <i>eccCb</i>_<i>1</i>::<i>tn</i>. B) Immuno-electron microscopy analysis of <i>M</i>. <i>tuberculosis</i> CDC1551, the <i>esx-5</i> mutant Mtb-<i>eccC</i>_<i>5</i>::<i>tn</i> and the mutant complemented with an integrative plasmid containing the complete <i>esx-5</i> region; pMV-<i>esx-5</i>. Cells were grown in liquid culture with 0.05% Tween-80 (lower row) or without Tween-80 (upper row) and labeled with a monoclonal antibody directed against PIM₆-LAM capsular glycolipids and a gold-labeled secondary antibody. C) Gold labeling of the different Mtb strains depicted in B was quantified by counting the number of gold particles per cell after growth in the presence or absence of Tween-80 (error bars indicate the standard deviation). D) The capsule morphology of wild-type <i>M</i>. <i>marinum</i> and <i>espG</i>_<i>5</i>::<i>tn</i> was analyzed after plunge freezing of bacteria grown in the absence of Tween-80 by cryo-electron microscopy. The black bar indicates the mycobacterial capsular layer. E) Reduced hydrophobicity of the <i>espG</i>_<i>5</i>::<i>tn</i> and <i>ppe10</i>::<i>tn</i> strains, measured as the OD of the aqueous phase after 1 OD of bacteria was incubated with 0.5% (v/v) Xylene in PBS for two hours. Statistical differences were calculated by GraphPad Prism software using one-way ANOVA and (Dunnett’s) multiple comparisons against a single control. ** = <i>p</i><0.01, n.s = not significant.</p

The <i>M</i>. <i>marinum ppe10</i>::tn mutant is attenuated in zebrafish embryo infections.

<p>A) <i>M</i>. <i>marinum</i> E11 wild type strain and the isogenic mutants <i>espG</i>_<i>5</i>::<i>tn</i>, <i>eccCb</i>_<i>1</i>::<i>tn</i> and <i>ppe10</i>::<i>tn</i>, as well as the complemented <i>ppe10</i>::<i>tn</i> strain (PPE10-C), were pre-cultured in liquid medium containing Tween-80. 50–100 CFUs of bacteria were injected in the bloodstream of zebrafish embryos at 28 hours post fertilization. Embryos were homogenized five days post infection and plated to establish the number of CFU per embryo. Three independent experiments of six embryos per group were performed and the data were pooled. B) Visualization of <i>M</i>. <i>marinum</i> infection of zebrafish embryos. Wild-type <i>M</i>. <i>marinum</i> or <i>ppe10</i>::<i>tn</i> containing the plasmid pSMT3::<i>mCherry</i> was injected in the bloodstream of zebrafish embryos as described above. After 5 days of infection the embryos were examined by brightfield (top) or fluorescence (bottom) microscopy. C) Quantification of fluorescence in infected zebrafish embryos. <i>M</i>. <i>marinum</i> wild type (closed symbols) and the <i>ppe10</i>::<i>tn</i> (open symbols) mutants expressing <i>mCherry</i> were injected in the bloodstream of zebrafish embryos 28 hours post fertilization. Images were acquired by fluorescence microscopy at day 5 (blue) and 7 (red) post infection and were analyzed for fluorescent intensity by dedicated software [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.ref043" target="_blank">43</a>]. Differences on day 5 and day 7 were analyzed by GraphPad Prism software using Mann-Whitney two-tailed test. * = <i>p</i><0.05, ** = <i>p</i><0.01, n.s. = not significant.</p

Both the <i>esx-5</i> and <i>ppe10</i> mutants have reduced hemolytic activity and show reduced ubiquitin co-localization upon infection of host cells.

<p>A) Contact-dependent hemolysis of red blood cells (RBCs) by <i>M</i>. <i>marinum</i>. <i>M</i>. marinum E11 wild-type cells and isogenic transposon mutants disrupted in <i>espG</i>_<i>5</i>, <i>eccCb</i>_<i>1</i> and <i>ppe10</i> and the complemented <i>ppe10</i> mutant were grown in the presence or absence of Tween-80. Subsequently, washed cells were used for the hemolysis assay. B) Quantification of differentiated THP-1 cells that were infected with <i>M</i>. <i>marinum</i>. Only cells gated as positive for co-localization of the bacteria with ubiquitin were chosen for further analysis (full data and statistics in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.s006" target="_blank">S6 Fig</a>). The <i>ppe10</i>::<i>tn</i> mutant showed similar levels of ubiquitin co-localization as <i>eccCb</i>_<i>1</i>::<i>tn</i> indicating that this mutant has no cytosolic access in the early stages of infection. C) Images obtained by imaging flow cytometry of wild-type <i>M</i>. <i>marinum</i> and <i>ppe10</i>::<i>tn</i> three hours after infection of differentiated THP-1 cells. Bacteria express <i>mCherry</i> (Red), while ubiquitin is visualized by FK-2 antibody against poly-ubiquitin (Green). D) Super-resolution confocal microscopy images of wild-type <i>M</i>. <i>marinum</i> and <i>ppe10</i>::<i>tn</i> illustrating differential bacterial clustering and ubiquitin labeling of bacteria. Bacteria express <i>mCherry</i> (Red), while ubiquitin is visualized by FK-2 antibody against poly-ubiquitin (Green). A 3-dimensional view of these images can be found in supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.s009" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005696#ppat.1005696.s010" target="_blank">S2</a> Movies.</p