Decreased Cell Wall Research Articles

MsMIOX2, encoding a MsbZIP53-activated myo-inositol oxygenase, enhances saline-alkali stress tolerance by regulating cell wall pectin and hemicellulose biosynthesis in alfalfa.

Alfalfa is one of the most widely cultivated forage crops worldwide. However, soil salinization restricts alfalfa growth and development and affects global productivity. The plant cell wall is the first barrier against various stresses. Therefore, elucidating the alterations in cell wall architecture is crucial for stress adaptation. This study aimed to clarify the impact of myo-inositol oxygenase 2 (MsMIOX2) on cell wall pectin and hemicellulose biosynthesis under saline-alkali stress and identify the upstream transcription factors that govern MsMIOX2. MsMIOX2 activation induced cell wall pectin and hemicellulose accumulation under saline-alkali stress. The effects of MsMIOX2 in saline-alkali tolerance were investigated by characterizing its overexpression and RNA interference lines. MsMIOX2 overexpression positively regulated the antioxidant system and photosynthesis in alfalfa under saline-alkali stress. MsMIOX2 exhibited myo-inositol oxygenase activity, which increased polysaccharide contents, facilitated pectin and hemicellulose biosynthesis, and extended the cell wall thickness. However, MsMIOX2 RNA interference decreased cell wall thickness and alleviated alfalfa saline-alkali stress tolerance. In addition, MsbZIP53 was identified as an upstream transcriptional MsMIOX2 regulator by yeast one-hybrid, electrophoretic mobility shift assay, dual-luciferase, and beta-glucuronidase assays. MsbZIP53 overexpression increased MsMIOX2 expression, elevated MIOX activity, reinforced the antioxidant system and photosynthesis, and increased saline-alkali stress tolerance in alfalfa. In conclusion, this study presents a novel perspective for elucidating the molecular mechanisms of saline-alkali stress tolerance in alfalfa and emphasizes the potential use of MsMIOX2 in alfalfa breeding.

A 3-component module maintains sepal flatness in Arabidopsis

As in origami, morphogenesis in living systems heavily relies on tissue curving and folding through the interplay between biochemical and biomechanical cues. By contrast, certain organs maintain their flat posture over several days. Here, we identified a pathway that is required for the maintenance of organ flatness, taking the sepal, the outermost floral organ, in Arabidopsis as a model system. Through genetic, cellular, and mechanical approaches, our results demonstrate that the global gene expression regulator VERNALIZATION INDEPENDENCE 4 (VIP4) fine-tunes the mechanical properties of sepal cell walls and maintains balanced growth on both sides of the sepals, mainly by orchestrating the distribution pattern of AUXIN RESPONSE FACTOR 3 (ARF3). vip4 mutation results in softer cell walls and faster cell growth on the adaxial sepal side, which eventually cause sepals to bend outward. Downstream of VIP4, ARF3 works through modulating auxin to downregulate pectin methylesterase VANGUARD1, resulting in decreased cell wall stiffness. Thus, our work unravels a 3-component module that relates hormonal patterns to organ curvature and actively maintains sepal flatness during its growth.

Defective pgsA contributes to increased membrane fluidity and cell wall thickening in Staphylococcus aureus with high-level daptomycin resistance.

The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to β-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA, on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance.

Alginate Oligosaccharides Alleviate Salt Stress in Rice Seedlings by Regulating Cell Wall Metabolism to Maintain Cell Wall Structure and Improve Lodging Resistance.

Salt stress is one of the major abiotic stresses that damage the structure and composition of cell walls. Alginate oligosaccharides (AOS) have been advocated to significantly improve plant stress tolerance. The metabolic mechanism by which AOS induces salt tolerance in rice cell walls remains unclear. Here, we report the impact of AOS foliar application on the cell wall composition of rice seedlings using the salt-tolerant rice variety FL478 and the salt-sensitive variety IR29. Data revealed that salt stress decreased biomass, stem basal width, stem breaking strength, and lodging resistance; however, it increased cell wall thickness. In leaves, exogenous AOS up-regulated the expression level of OSCESA8, increased abscisic acid (ABA) and brassinosteroids (BR) content, and increased β-galacturonic activity, polygalacturonase activity, xylanase activity, laccase activity, biomass, and cellulose content. Moreover, AOS down-regulated the expression levels of OSMYB46 and OSIRX10 and decreased cell wall hemicellulose, pectin, and lignin content to maintain cell wall stability under salt stress. In stems, AOS increased phenylalamine ammonia-lyase and tyrosine ammonia-lyase activities, while decreasing cellulase, laccase, and β-glucanase activities. Furthermore, AOS improved the biomass and stem basal width and also enhanced the cellulose, pectin, and lignin content of the stem, As a result, increased resistance to stem breakage strength and alleviated salt stress-induced damage, thus enhancing the lodging resistance. Under salt stress, AOS regulates phytohormones and modifies cellulose, hemicellulose, lignin, and pectin metabolism to maintain cell wall structure and improve stem resistance to lodging. This study aims to alleviate salt stress damage to rice cell walls, enhance resistance to lodging, and improve salt tolerance in rice by exogenous application of AOS.

Bifunctional transcription factors SlERF.H5 and H7 activate cell wall and repress gibberellin biosynthesis genes in tomato via a conserved motif

The plant cell wall is a dynamic structure that plays an essential role in development, but the mechanism regulating cell wall formation remains poorly understood. We demonstrate that two transcription factors, SlERF.H5 and SlERF.H7, control cell wall formation and tomato fruit firmness in an additive manner. Knockout of SlERF.H5, SlERF.H7, or both genes decreased cell wall thickness, firmness, and cellulose contents in fruits during early development, especially in double-knockout lines. Overexpressing either gene resulted in thicker cell walls and greater fruit firmness with elevated cellulose levels in fruits but severely dwarf plants with lower gibberellin contents. We further identified that SlERF.H5 and SlERF.H7 activate the cellulose biosynthesis gene SlCESA3 but repress the gibberellin biosynthesis gene GA20ox1. Moreover, we identified a conserved LPL motif in these ERFs responsible for their activities as transcriptional activators and repressors, providing insight into how bifunctional transcription factors modulate distinct developmental processes.

Leaf diffusional capacity largely contributes to the reduced photosynthesis in rice plants under magnesium deficiency

Numerous studies have clarified the impacts of magnesium (Mg) on leaf photosynthesis from the perspectives of protein synthesis, enzymes activation and carbohydrate partitioning. However, it still remains largely unknown how stomatal and mesophyll conductances (gs and gm, respectively) are regulated by Mg. In the present study, leaf gas exchanges, leaf hydraulic parameters, leaf structural traits and cell wall composition were examined in rice plants grown under high and low Mg treatments to elucidate the impacts of Mg on gs and gm. Our results showed that reduction of leaf photosynthesis under Mg deficiency was mainly caused by the decreased gm, followed by reduced leaf biochemical capacity and gs, and leaf outside-xylem hydraulic conductance (Kox) was the major factor restricting gs under Mg deficiency. Moreover, increased leaf hemicellulose, lignin and pectin contents and decreased cell wall effective porosity were observed in low Mg plants relative to high Mg plants. These results suggest that Kox and cell wall composition play important roles in regulating gs and gm, respectively, in rice plants under Mg shortages.

Freezing pre-treatment improves radio frequency explosion puffing (RFEP) quality by altering the cellular structure of purple sweet potato [Ipomoea batatas (L) Lam.

Radio frequency explosion puffing (RFEP) is a novel oil-free puffing technique used to produce crispy textured and nutritious puffed snacks. This study aimed to investigate the effects of freezing at different temperatures (−20 °C, −40 °C, −80 °C) for14 h and freezing times (1 and 2 times) on the cellular structure of purple sweet potato and the quality of RFEP chips. The analysis of cell microstructure, conductivity, and rheology revealed that higher freezing temperatures and more freezing times resulted in increased damage to the cellular structure, leading to greater cell membrane permeability and decreased cell wall stiffness. However, excessive damage to cellular structure caused tissue structure to collapse. Compared with the control group (4 °C), the RFEP sample pre-frozen once at −40 °C had a 47.13 % increase in puffing ratio and a 61.93 % increase in crispness, while hardness decreased by 23.44 % (p < 0.05). There was no significant change in anthocyanin retention or color difference. X-ray microtomography demonstrated that the RFEP sample pre-frozen once at −40 °C exhibited a more homogeneous morphology and uniform pore distribution, resulting in the highest overall acceptability. In conclusion, freezing pre-treatment before RFEP can significantly enhance the puffing quality, making this an effective method for preparing oil-free puffing products for fruits and vegetables.

A newly discovered glycosyltransferase gene UGT88A1 affects growth and polysaccharide synthesis of Grifola frondosa

Grifolafrodosa polysaccharides, especially β-D-glucans, possess significant anti-tumor, antioxidant and immunostimulatory activities. However, the synthesis mechanism remains to be elucidated. A newly discovered glycosyltransferase UGT88A1 was found to extend glucan chains in vitro. However, the role of UGT88A1 in the growth and polysaccharide synthesis of G. frondosa in vivo remains unclear. In this study, the overexpression of UGT88A1 improved mycelial growth, increased polysaccharide production, and decreased cell wall pressure sensitivity. Biomass and polysaccharide production decreased in the silenced strain, and the pressure sensitivity of the cell wall increased. Overexpression and silencing of UGT88A1 both affected the monosaccharide composition and surface morphology of G. frondosa polysaccharides and influenced the antioxidant activity of polysaccharides from different strains. The messenger RNA expression of glucan synthase (GLS), UTP-glucose-1-phosphate uridylyltransferase (UGP), and UDP-xylose-4-epimerase (UXE) related to polysaccharide synthesis, and genes related to cell wall integrity increased in the overexpression strain. Overall, our study indicates that UGT88A1 plays an important role in the growth, stress, and polysaccharide synthesis of G. frondosa, providing a reference for exploring the pathway of polysaccharide synthesis and metabolic regulation.Key points•UGT88A1 plays an important role in the growth, stress response, and polysaccharide synthesis in G. frondosa.•UGT88A1 affected the monosaccharide composition, surface morphology and antioxidant activity of G. frondosa polysaccharides.•UGT88A1 regulated the mRNA expression of genes related to polysaccharide synthesis and cell wall integrity.

A critical concentration of magainin 2 is required to decrease cell stiffness but not to decrease cell wall rigidity in Staphylococcus epidermidis

A critical concentration of magainin 2 is required to decrease cell stiffness but not to decrease cell wall rigidity in Staphylococcus epidermidis

Bacterial eradication by a low-energy pulsed electron beam generator

Low-energy electron beams (LEEB) are a safe and practical sterilization solution for in-line industrial applications, such as sterilizing medical products. However, their low dose rate induces product degradation, and the limited maximal energy prohibits high-throughput applications. To address this, we developed a low-energy ‘pulsed’ electron beam generator (LEPEB) and evaluated its efficacy and mechanism of action. Bacillus pumilus vegetative cells and spores were irradiated with a 250 keV LEPEB system at a 100 Hz pulse repetition frequency and a pulse duration of only 10 ns. This produced highly efficient bacterial inactivation at a rate of >6 log10, the level required for sterilization in industrial applications, with only two pulses for vegetative bacteria (20 ms) and eight pulses for spores (80 ms). LEPEB induced no morphological or structural defects, but decreased cell wall hydrophobicity in vegetative cells, which may inhibit biofilm formation. Single- and double-strand DNA breaks and pyrimidine dimer formation were also observed, likely causing cell death. Together, the unique combination of high dose rate and nanosecond delivery of LEPEB enable effective and high-throughput bacterial eradication for direct integration into production lines in a wide range of industrial applications.

Insect fungal pathogens secrete a cell wall-associated glucanase that acts to help avoid recognition by the host immune system.

Fungal insect pathogens have evolved diverse mechanisms to evade host immune recognition and defense responses. However, identification of fungal factors involved in host immune evasion during cuticular penetration and subsequent hemocoel colonization remains limited. Here, we report that the entomopathogenic fungus Beauveria bassiana expresses an endo-β-1,3-glucanase (BbEng1) that functions in helping cells evade insect immune recognition/ responses. BbEng1 was specifically expressed during infection, in response to host cuticle and hemolymph, and in the presence of osmotic or oxidative stress. BbEng1 was localized to the fungal cell surface/ cell wall, where it acts to remodel the cell wall pathogen associated molecular patterns (PAMPs) that can trigger host defenses, thus facilitating fungal cell evasion of host immune defenses. BbEng1 was secreted where it could bind to fungal cells. Cell wall β-1,3-glucan levels were unchanged in ΔBbEng1 cells derived from in vitro growth media, but was elevated in hyphal bodies, whereas glucan levels were reduced in most cell types derived from the BbEng1 overexpressing strain (BbEng1OE). The BbEng1OE strain proliferated more rapidly in the host hemocoel and displayed higher virulence as compared to the wild type parent. Overexpression of their respective Eng1 homologs or of BbEng1 in the insect fungal pathogens, Metarhizium robertsii and M. acridum also resulted in increased virulence. Our data support a mechanism by which BbEng1 helps the fungal pathogen to evade host immune surveillance by decreasing cell wall glucan PAMPs, promoting successful fungal mycosis.

Manipulating microRNA miR408 enhances both biomass yield and saccharification efficiency in poplar

The conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis, with associated negative growth impacts. Here we demonstrate, for both laboratory- and field-grown plants, that expression of Pag-miR408 in poplar (Populus alba × P. glandulosa) significantly enhances saccharification, with no requirement for acid-pretreatment, while promoting plant growth. The overexpression plants show increased accessibility of cell walls to cellulase and scaffoldin cellulose-binding modules. Conversely, Pag-miR408 loss-of-function poplar shows decreased cell wall accessibility. Overexpression of Pag-miR408 targets three Pag-LACCASES, delays lignification, and modestly reduces lignin content, S/G ratio and degree of lignin polymerization. Meanwhile, the LACCASE loss of function mutants exhibit significantly increased growth and cell wall accessibility in xylem. Our study shows how Pag-miR408 regulates lignification and secondary growth, and suggest an effective approach towards enhancing biomass yield and saccharification efficiency in a major bioenergy crop.

Combined Transcriptome and Metabolome Analysis Reveals That the Potent Antifungal Pyrylium Salt Inhibits Mitochondrial Complex I in Candida albicans.

Based on the structural modification of SM21, xy12, a new pyrylium salt derivative with enhanced antifungal activities, was synthesized. The MICs (MIC90) of xy12 against Candida albicans ranged from 0.125 to 0.25 μg/mL, about 2-fold lower than those of SM21. In addition, xy12 inhibited hypha and biofilm formation in C. albicans in a dose-dependent manner. A total of 3,454 differentially expressed genes and 260 differential metabolites were identified in the xy12-treated C. albicans by RNA-seq and non-targeted metabolomics. By integrating KEGG pathway enrichment analysis, we found that inhibition of oxidative phosphorylation was the important antifungal mechanism of action of xy12. Electron transport through mitochondrial respiratory complexes I to IV is the common process of oxidative phosphorylation. Compared with the sensitivity of the wild-type SC5314 to xy12, decreased sensitivities in mitochondrial complex I (CI)-deficient mutants and increased sensitivities in mitochondrial complex III- and IV-deficient mutants suggested that the antifungal effects of xy12 were dependent on CI. Consistently, xy12 exhibited antagonism with rotenone, an inhibitor of CI, and significantly inhibited the expression and activity of CI. Meanwhile, the phenotypes in the xy12-treated C. albicans were similar to those in the CI-deficient mutants, such as decreased ATP production, reduced mitochondrial membrane potential, loss of mitochondrial DNA, inability to utilize nonfermentative carbon sources, and decreased cell wall N-linked mannoproteins. Collectively, our results revealed that the pyrylium salt xy12 could constrain oxidative phosphorylation by inhibiting mitochondrial complex I in C. albicans, providing a novel lead compound for the development of mitochondria-targeted antifungal drugs. IMPORTANCE The development of new antifungal drugs is critical for solving the problem of antifungal resistance and expanding the limited variety of clinical antifungal drugs. Based on the modification of the pyrylium salt SM21, a new lead compound, xy12, was synthesized which was effective against Candida species both in vitro and in vivo. In this study, conjoined analysis of the transcriptome and metabolome elucidated the antifungal mechanism of action of xy12, which inhibited the activity of mitochondrial complex I in C. albicans. Targeting fungi-specific mitochondrial complex proteins has been reported as a promising antifungal strategy. Our study provided a new lead compound for targeting C. albicans mitochondrial complex I, which could be beneficial for discovering novel antifungal drugs.

Decrease in hemicellulose content and its retention of iron contributes to phosphorus deficiency alleviated iron deficiency in Arabidopsis thaliana

The physiological and molecular mechanisms between phosphorus (P) and iron (Fe) interactions are still elusive although they have been extensively investigated. In this study, we uncovered that limiting P supply could alleviate Fe deficiency in Arabidopsis (Col-0). Under Fe deficiency, P deficiency (-Fe-P) decreased cell wall Fe accumulation in root, but elevated Fe accumulation in the shoot, implying that the reduced Fe retention in the root cell wall may contribute to the P-deficiency-alleviated Fe deficiency in the shoot. On the other hand, increasing P supply could mimic the degree of Fe deficiency in terms of the expressions of genes induced after Fe deficient treatment. The components of the root cell wall showed that there was no distinction in the pectin content and the Fe retention in pectin between -Fe and -Fe-P treatments, while hemicellulose 1 content and Fe retained in it were decreased significantly in -Fe-P treatment as compared with -Fe treatment. The time-course experiment showed that decreasing cell wall retained Fe was mainly from the corresponding decrease in hemicellulose 1 retained Fe. Furthermore, the up-regulation of IRT1 expression in -Fe-P was obviously lower than -Fe. All these suggest that the P deficiency-induced decrease of hemicellulose 1 component leads to reutilization of root cell wall Fe and improvement of Fe nutrition in shoot in Fe deficient Arabidopsis. Our results provide a novel explanation of the interplay between Fe and P in Arabidopsis.

Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA–MurG interaction

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.

Modification of Xyloglucan Metabolism during a Decrease in Cell Wall Extensibility in 1-Aminocyclopropane-1-Carboxylic Acid-Treated Azuki Bean Epicotyls.

The exogenous application of ethylene or 1-aminocyclopropane-1-carboxylic acid (ACC), the biosynthetic precursor for ethylene, to plants decreases the capacity of the cell wall to extend, thereby inhibiting stem elongation. In this study, the mechanism by which the extensibility of cell walls decreases in ACC-treated azuki bean epicotyls was studied. ACC decreased the total extensibility of cell walls, and such a decrease was due to the decrease in irreversible extensibility. ACC increased the molecular mass of xyloglucans but decreased the activity of xyloglucan-degrading enzymes. The expression of VaXTHS4, which only exhibits hydrolase activity toward xyloglucans, was downregulated by ACC treatment, whereas that of VaXTH1 or VaXTH2, which exhibits only transglucosylase activity toward xyloglucans, was not affected by ACC treatment. The suppression of xyloglucan-degrading activity by downregulating VaXTHS4 expression may be responsible for the increase in the molecular mass of xyloglucan. Our results suggest that the modification of xyloglucan metabolism is necessary to decrease cell wall extensibility, thereby inhibiting the elongation growth of epicotyls in ACC-treated azuki bean seedlings.

Flagging defensive roles of carbohydrate-active enzymes (CAZymes) and carbohydrates during Puccinia triticina-wheat interactions

Puccinia triticina (Pt) infects wheat, leading to colonization of leaf tissues, pustule formation on the leaf and eventually yield loss. Fungicides and breeding techniques are used to manage Pt infection in wheat. However, there is a growing need for biochemical studies to identify critical metabolites and proteins that contribute to plant susceptibility or resistance against Pt. The current study investigated defensive role of non-structural, structural carbohydrates, and CAZymes during Pt-wheat interaction at 1, 7, and 14 days post inoculation (dpi). The results revealed that glucose, total reducing sugars and sucrose were significantly decreased by more than 50% in Pt inoculated susceptible Thatcher (IS) plants. Sucrose concentrations decreased by 0.80 and 0.93 fold in IS compared to control sample (CS) at 1 and 7 dpi. Pt inoculated resistant Thatcher + Lr9 (IR) plants and control (CR) did not display differences in soluble carbohydrate concentrations. In addition, exoglucanase, endoglucanase, pectinase and carbohydrate esterase activities were significantly higher at 1 or 7 dpi in IS compared to IR plants. The cellulose-content was 29% in IR samples compared to 15% in CR samples at 14 dpi. Additionally, hemicellulose increased by 2% in IR at 14 dpi. The cell wall reinforcements in resistant plants were supported by Fourier transformation infrared results that revealed that IR holocellulose content was not affected by Pt infection, but IS samples holocellulose decreased at 7 dpi. In conclusion, increased activities of CAZymes, decreased cell wall holocellulose content, and reduced soluble carbohydrates were susceptibility indicators.

Increasing extracellular cellulase activity of the recombinant Saccharomyces cerevisiae by engineering cell wall-related proteins for improved consolidated processing of carbon neutral lignocellulosic biomass

Sustainable bioproduction usingcarbon neutral feedstocks, especially lignocellulosic biomass, has attracted increasing attention due to concern over climate change and carbon reduction. Consolidated bioprocessing (CBP) of lignocellulosic biomass using recombinantyeast of Saccharomyces cerevisiaeis a promising strategy forlignocellulosic biorefinery. However, the economic viability is restricted by low enzyme secretion levels.For more efficient CBP, MIG1spsc01isolated from the industrial yeast which encodes the glucose repression regulator derivative was overexpressed. Increased extracellular cellobiohydrolase (CBH) activity was observed with unexpectedly decreased cell wall integrity. Further studies revealed that disruption ofCWP2, YGP1, andUTH1,which are functionally related toMIG1spsc01, also enhanced CBH secretion. Subsequently, improved cellulase production was achieved by simultaneous disruption ofYGP1and overexpression ofSED5, which remarkably increased extracellular CBH activity of 2.2-fold over the control strain. These results provide a novel strategy to improve the CBP yeast for bioconversion of carbon neutral biomass.

Calcium decreases cell wall swelling in sweet cherry fruit

Swelling of epidermal cell walls decreases cell-to-cell adhesion and increases cracking susceptibility in sweet cherry. Ca is suggested to decrease cracking susceptibility by crosslinking of cell wall components and, possibly, by decreasing swelling. The objective is to test this hypothesis. The effect of Ca on swelling of anticlinal epidermal cell walls was quantified microscopically in vivo using excised skin sections and in vitro using extracted cell walls. After removal of turgor, cell wall thickness increased. Incubation in CaCl2 decreased cell wall thickness up to 3 mM CaCl2. At higher concentrations thickness remained constant. Decreased cell wall swelling in vivo also occurred with other salts of divalent and trivalent cations, but not with those of monovalent cations. Decreased swelling was due to the Ca cation, the anions had no effect. Ca also decreased swelling of cell walls that were already swollen. CaCl2 also decreased swelling of extracted cell walls in vitro. There was no effect on swelling pressure. The effect on swelling increased as the CaCl2 concentration increased. Chlorides of divalent and trivalent cations, but not those of monovalent cations decreased swelling in vitro. The decrease in swelling among the divalent cations was linearly related to the radius of the cation. The results indicate that Ca decreases cracking susceptibility by decreasing swelling.

Mycobacterium tuberculosis Rv0309 Dampens the Inflammatory Response and Enhances Mycobacterial Survival.

To reveal functions of novel Mycobacterium tuberculosis (M. tb) proteins responsible for modulating host innate immunity is essential to elucidation of mycobacterial pathogenesis. In this study, we aimed to identify the role of a putative protein Rv0309 encoded within RD8 of M. tb genome in inhibiting the host inflammatory response and the underlying mechanism, using in-vitro and in-vivo experiments. A recombinant M. smegmatis strain Ms_rv0309 expressing Rv0309 and a mutant Bacillus Calmette-Guérin (BCG)ΔRS01790 strain with deletion of BCG_RS01790, 100% homologue of Rv0309 in BCG, were constructed. Rv0309 was found to localize in the cell wall and be able to decrease cell wall permeability. Purified recombinant rRv0309 protein inhibited lipopolysaccharide-induced IL-6 release in RAW264.7 cells. BCG_RS01790 in BCG or Rv0309 in Ms_rv0309 strain greatly inhibited production of IL-6, IL-1β, and TNF-α in RAW264.7 cells. Similarly, BCGΔRS01790 strongly induced expression of these cytokines compared with wild-type BCG and complement strain, cBCGΔRS01790::RS01790. Further BCG_RS01790 or Rv0309 suppressed cytokine production through NF-κB p65/IκBα and MAPK ERK/JNK signaling. Importantly, BCG_RS01790 in BCG and Rv0309 in Ms_rv0309 strain enhanced mycobacterial survival in macrophages. Mice infected with BCGΔRS01790 exhibited high levels of IFN-γ, TNF-α and IL-1β, and large numbers of neutrophils and lymphocytes in the early stage, and minimal lung bacterial load and inflammatory damage in late stage of the experiment. In conclusion, the cell wall protein Rv0309 or BCG_RS01790 enhanced mycobacterial intracellular survival after infection likely through inhibition of the pro-inflammatory response and decrease of bacterial cell wall permeability, thereby contributing to mycobacterial pathogenesis.