Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation

Vitikainen, Marika; Hyyryläinen, Hanne‐Leena; Kivimäki, Anna; Kontinen, Vesa P.; Sarvas, Matti

doi:10.1111/j.1365-2672.2005.02572.x

Cited by 55 publications

(52 citation statements)

References 47 publications

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“…6). In this regard, we note that the prsA homolog in Bacillus subtilis has been extensively studied, and its dysregulated expression, resulting in either increased or decreased prsA transcription, impairs the secretion and extracellular processing of other proteins (23)(24)(25). SpeB is well known to be a critical virulence factor for severe invasive episodes of GAS infection, and its genetic inactivation significantly decreases virulence and tissue destruction (16)(17)(18)(19)(20)(21)(22).…”

Section: Discussionmentioning

confidence: 99%

“…This gene encodes a peptidyl-prolyl isomerase required for normal maturation and enzymatic activity of streptococcal pyrogenic exotoxin B (SpeB) (15), a critical broad-spectrum secreted protease virulence factor involved in the degradation of extracellular matrix proteins, inactivation of innate immune molecules, and destruction of host tissue (16)(17)(18)(19)(20)(21)(22). Although control of prsA transcription is not well understood in GAS (15), dysregulation of prsA homologs (either increased or decreased expression of prsA) in other bacterial species has been shown to detrimentally alter the maturation of secreted proteins that are dependent on PrsA for extracellular processing (23)(24)(25). Also, SpeB is abundantly expressed in situ in infected human tissues (26).…”

Section: Wild-type Strain Mgas315 Causes Significantly More Extensivementioning

confidence: 99%

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Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis

Olsen

Sitkiewicz

Ayeras

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

141

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Single-nucleotide changes are the most common cause of natural genetic variation among members of the same species, but there is remarkably little information bearing on how they alter bacterial virulence. We recently discovered a single-nucleotide mutation in the group A Streptococcus genome that is epidemiologically associated with decreased human necrotizing fasciitis ("flesh-eating disease"). Working from this clinical observation, we find that wild-type mtsR function is required for group A Streptococcus to cause necrotizing fasciitis in mice and nonhuman primates. Expression microarray analysis revealed that mtsR inactivation results in overexpression of PrsA, a chaperonin involved in posttranslational maturation of SpeB, an extracellular cysteine protease. Isogenic mutant strains that overexpress prsA or lack speB had decreased secreted protease activity in vivo and recapitulated the necrotizing fasciitis-negative phenotype of the ΔmtsR mutant strain in mice and monkeys. mtsR inactivation results in increased PrsA expression, which in turn causes decreased SpeB secreted protease activity and reduced necrotizing fasciitis capacity. Thus, a naturally occurring single-nucleotide mutation dramatically alters virulence by dysregulating a multiple gene virulence axis. Our discovery has broad implications for the confluence of population genomics and molecular pathogenesis research.group A streptococcus | invasive infection | molecular epidemiology of strain genotype patient phenotype relationships | nonhuman primate S ingle-nucleotide mutations are the most abundant cause of genetic variation among members of the same species (1, 2). However, in striking contrast to humans, who have been studied extensively, our understanding of how naturally occurring singlenucleotide mutations alter bacterial phenotypes is rudimentary. Most prokaryotic pathogenesis research efforts have focused intensively on large regions of genetic difference, such as pathogenicity islands and prophages. Thus, there is little information that directly bears on the relationship between particular singlenucleotide changes, their direct or indirect effect on virulence factor expression, and the manifestation of medically important traits such as strain virulence and infection specificity.Recently, we have investigated the molecular genomic landscape of infection phenotype-strain genotype relationships in human patients at the nucleotide level in group A Streptococcus (GAS), a bacterial pathogen that is a major cause of human morbidity and mortality worldwide (1, 3-5). These studies were made possible by the availability of the 1.9-Mb genome sequences of 12 GAS strains cultured from patients with welldefined clinical syndromes such as pharyngitis, acute rheumatic fever, and necrotizing fasciitis (also known as "flesh-eating" disease) (1, 5). The core genome of strains of distinct M protein serotype differed, on average, by 14,475 SNPs (1, 3). In contrast, strains with the same M protein serotype were far less variable, differing overall by less...

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Section: Discussionmentioning

confidence: 99%

Section: Wild-type Strain Mgas315 Causes Significantly More Extensivementioning

confidence: 99%

Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis

Olsen

Sitkiewicz

Ayeras

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

141

View full text Add to dashboard Cite

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“…Since a mutation of CssRS will effectively prevent the induction of HtrA and HtrB (7), deletion of these two induced proteases separately is unlikely to improve secretion of ␤-toxoid. In studies performed by Vitikainen et al (33), it was also observed that downregulation or mutation of HtrA and/or HtrB proteases does not improve secretion but instead induces severe stress in the cells, resulting in poor growth and generally lower secretion yields. However, Since ykoJ was strongly overexpressed in our study, we tested whether this protein itself influences the efficiency of secretion of ␤-toxoid.…”

Section: Resultsmentioning

confidence: 99%

“…Expression of chaperones and proteases can be altered (33), the charge of the cell wall or the secreted protein can be adapted (28), and the availability of divalent metal ions can accelerate folding of the proteins (27). However, such measures usually improve secretion by only a factor of 1.5 to 3.…”

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confidence: 99%

Changing a Single Amino Acid in Clostridium perfringens β-Toxin Affects the Efficiency of Heterologous Secretion by Bacillus subtilis

Nijland

Heerlien

Hamoen

et al. 2007

Appl Environ Microbiol

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Achieving efficient heterologous protein production and secretion by Bacillus subtilis is an attractive prospect, although often disappointingly low yields are reached. The expression of detoxified Clostridium perfringens ␤-toxin (␤-toxoid) is exemplary for this. Although ␤-toxin can be efficiently expressed and secreted by Bacillus subtilis, the genetically detoxified, and industrially interesting, ␤-toxoid variant is difficult to obtain in high amounts. To optimize the expression of this putative vaccine component, we studied the differences in the global gene regulation responses of B. subtilis to overproduction of either ␤-toxin or ␤-toxoid by transcriptomics. A clear difference was the upregulation of the CssRS regulon, known to be induced upon secretion stress, when ␤-toxoid is produced. YkoJ, a protein of unknown function, was also upregulated, and we show that its expression is dependent on cssS. We then focused on the heterologous protein itself and found that the major secretion bottleneck can be traced back to a single amino acid substitution between the ␤-toxin and the ␤-toxoid, which results in the rapid degradation of ␤-toxoid following secretion across the cytoplasmic membrane. In contrast to ␤-toxin, ␤-toxoid protein is more prone to degradation directly after secretion, most likely due to poor folding characteristics introduced with point mutations. Our results show that although the host can be adapted in many ways, the intrinsic properties of a heterologous protein can play a decisive role when optimizing heterologous protein production.

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“…Mutations in dlt increase the density of negative charge in the cell wall matrix. In addition to PrsA3, some other mutant and heterologous proteins are also stabilized and their secretion improved when Dlt is inactivated (Craynest et al, 2003;Hyyryläinen et al, 2000;Thwaite et al, 2002;Vitikainen et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

The density of negative charge in the cell wall influences two-component signal transduction in Bacillus subtilis

et al. 2007

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The Dlt system modulates the density of negative charge in the cell wall of Gram-positive bacteria by substituting anionic polymers (wall and lipoteichoic acids) with D-alanine. The htrA and htrB genes, regulated by the CssRS two-component system (TCS) and encoding membrane-associated protein quality control proteases, were expressed at strongly decreased levels in a mutant with defective Dlt (dltD : : miniTn10) as compared to the dlt + wild-type strain under a secretion stress condition (hypersecretion of AmyQ a-amylase). The level of HtrA protein in the extracellular proteome of the dltD mutant was decreased consistently. Expression from the promoter of the liaIHGFSR (yvqIHGFEC) operon (P liaI ) is dependent on the LiaRS TCS. The Dlt defect increased the expression from P liaI under two stress conditions, AmyQ hypersecretion and treatment with a cationic antimicrobial peptide (LL-37), but decreased the expression in vancomycin-treated cells. Furthermore, Dlt inactivation enhanced the expression of the YxdJK-regulated yxdL gene in LL-37-treated cells. The increased net negative charge of the cell wall seems to cause varied and opposite effects on the expression of CssRS-, LiaRS-and YxdJK-regulated genes under different stress conditions. The results suggest that TCSs which sense misfolded proteins or peptides are modulated by the density of negative charge in the cell wall. The density of negative charge on the outer surface of the cell membrane did not have a similar effect on TCSs.

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Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation

Cited by 55 publications

References 47 publications

Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis

Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis

Changing a Single Amino Acid in Clostridium perfringens β-Toxin Affects the Efficiency of Heterologous Secretion by Bacillus subtilis

The density of negative charge in the cell wall influences two-component signal transduction in Bacillus subtilis

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