Is colonoscopy necessary in cases of infection by Streptococcus bovis biotype II?

The association of colorectal neoplasia (CRN) with Streptococcus bovis biotype I (SBI) infection is well recognized. However, this is not the case for Streptococcus bovis biotype II (SBII). We conducted this study in order to analyze the relationship between SBII and CRN. We analyzed all cases of bacteremia due to SBI (n = 99) and SBII (n = 36) diagnosed in our hospital (during the period 1988-2011) that were followed up with colonoscopy. In addition, we reviewed the literature (during the period 1982-2011) to select all cases of infection of SB that had undergone colonoscopy or other adequate form of colorectal examination. A multivariate analysis was performed to detect CRN risk factors in patients infected with SB. From the 223 cases of SB infection included in the analysis (135 from our institution and 88 from the literature review), 159 were due to SBI and 64 were caused by SBII. As compared with SBI, the SBII cases had a lower frequency of CRN (27 % vs. 67 %, p <0.001), advanced adenomas (8 % vs. 29 %, p <0.01), and carcinomas (6 % vs. 21 %, p <0.01). In a multivariate analysis, and after adjusting for age, sex, type of infection, and biotype, SBII infection was not associated with CRN: odds ratio (OR) = 0.17; 95 % confidence interval (CI) = 0.09 to 0.33. The only factor independently associated with CRN was SBI infection: OR = 5.7; 95 % CI = 3.0 to 10.9. The prevalence of CRN in patients infected with SBII is significantly lower than patients with SBI and does not appear to be higher than the CRN prevalence among the general population.

Iron metabolism in the pathogenesis of iron-induced kidney injury.

In the past 8 years, there has been renewed interest in the role of iron in both acute kidney injury (AKI) and chronic kidney disease (CKD). In patients with kidney diseases, renal tubules are exposed to a high concentration of iron owing to increased glomerular filtration of iron and iron-containing proteins, including haemoglobin, transferrin and neutrophil gelatinase-associated lipocalin (NGAL). Levels of intracellular catalytic iron may increase when glomerular and renal tubular cells are injured. Reducing the excessive luminal or intracellular levels of iron in the kidney could be a promising approach to treat AKI and CKD. Understanding the role of iron in kidney injury and as a therapeutic target requires insight into the mechanisms of iron metabolism in the kidney, the role of endogenous proteins involved in iron chelation and transport, including hepcidin, NGAL, the NGAL receptor and divalent metal transporter 1, and iron-induced toxic effects. This Review summarizes emerging knowledge, which suggests that complex mechanisms of iron metabolism exist in the kidney, modulated directly or indirectly by cellular iron content, inflammation, ischaemia and oxidative stress. The potential exists for prevention and treatment of iron-induced kidney injury by customized iron removal or relocation, aided by detailed insight into the underlying pathological mechanisms.

Overweight impairs efficacy of iron supplementation in iron-deficient South African children: a randomized controlled intervention.

BACKGROUND: Many countries in the nutrition transition have high rates of iron deficiency (ID) and overweight (OW). ID is more common in OW children; this may be due to adiposity-related inflammation reducing iron absorption. OBJECTIVE: We investigated whether weight status predicts response to oral iron supplementation in ID South African children. DESIGN: A placebo-controlled trial of oral iron supplementation (50 mg, 4 × weeks for 8.5 months) was done in ID 6- to 11-year-old children (n=321); 28% were OW or obese. BMI-for-age z-scores (BAZ), hepcidin (in a sub-sample), hemoglobin, serum ferritin (SF), transferrin receptor (TfR), zinc protoporphyrin (ZnPP) and C-reactive protein (CRP) were measured; body iron was calculated from the SF to TfR ratio. RESULTS: At baseline, BAZ correlated with CRP (r=0.201, P<0.001) and CRP correlated with hepcidin (r=0.384, P<0.001). Normal weight children supplemented with iron had significantly lower TfR concentrations at endpoint than the OW children supplemented with iron and the children receiving placebo. Higher BAZ predicted higher TfR (ß=0.232, P<0.001) and lower body iron (ß=-0.090, P=0.016) at endpoint, and increased the odds ratio (OR) for remaining ID at endpoint in both the iron and placebo groups (iron: OR 2.31, 95% CI: 1.13, 4.73; placebo: OR 1.78, 95% CI: 1.09, 2.91). In the children supplemented with iron, baseline hepcidin and BAZ were significant predictors of endpoint TfR, with a trend towards a hepcidin × BAZ interaction (P=0.058). CONCLUSION: South African children with high BAZ have a two-fold higher risk of remaining ID after iron supplementation. This may be due to their higher hepcidin concentrations reducing iron absorption. Thus, the current surge in OW in rapidly developing countries may undercut efforts to control anemia in vulnerable groups. The trial is registered at clinicaltrials.gov as NCT01092377.

Serum ferritin levels are associated with vascular damage in patients with nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Increased ferritin and body iron stores are frequently observed in nonalcoholic fatty liver disease (NAFLD), associated with heightened susceptibility to vascular damage. Conflicting data have been reported on the role of iron in atherosclerosis, with recent data suggesting that excess iron induces vascular damage by increasing levels of the hormone hepcidin, which would determine iron trapping into macrophages, oxidative stress, and promotion of transformation into foam cells. Aim of this study was to investigate the relationship between iron status and cardiovascular damage in NAFLD. METHODS AND RESULTS: Vascular damage was evaluated by common carotid arteries intima-media thickness (CC-IMT) measurement and plaque detection by ecocolor-doppler ultrasonography in 506 patients with clinical and ultrasonographic diagnosis of NAFLD, hemochromatosis gene (HFE) mutations by restriction analysis in 342 patients. Serum hepcidin-25 was measured by time-of-flight mass spectrometry in 143 patients. At multivariate analysis CC-IMT was associated with systolic blood pressure, glucose, LDL cholesterol, abdominal circumference, age, and ferritin (p=0.048). Carotid plaques were independently associated with age, ferritin, glucose, and hypertension. Ferritin reflected iron stores and metabolic syndrome components, but not inflammation or liver damage. Hyperferritinemia was associated with increased vascular damage only in patients with HFE genotypes associated with hepcidin upregulation by iron stores (p<0.0001), and serum hepcidin-25 was independently associated with carotid plaques (p=0.05). CONCLUSION: Ferritin levels, reflecting iron stores, are independent predictors of vascular damage in NAFLD. The mechanism may involve upregulation of hepcidin by increased iron stores in patients not carrying HFE mutations, and iron compartmentalization into macrophages.

Engineering of quorum-sensing systems for improved production of alkaline protease by Bacillus subtilis.

AIM: Engineering of Rap-Phr quorum-sensing systems of Bacillus subtilis and subsequent evaluation of the transcription of the aprE gene, encoding a major extracellular alkaline protease. METHODS AND RESULTS: Addition of synthetic Phr pentapeptides to the growth medium, or overproduction of pre-Phr peptides, slightly improved the transcription of the aprE gene in B. subtilis. Disruption of certain rap genes similarly improved the transcription of the aprE gene. The production of extracellular proteolytic enzymes was increased when the rapA mutation was combined with a degU32 (Hy) mutation for hyper-secretion. CONCLUSIONS: Certain Rap-Phr systems of B. subtilis seem to suppress extracellular AprE production. Although this may be an important feature under natural conditions, repression of AprE production by these systems is not desirable under fermentation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Although the levels of aprE transcriptional increase in this study are moderate, engineering of Rap-Phr systems may be used to improve the yield of Bacillus strains that are used for the production of the extracellular protease AprE, or Bacillus strains that use of the aprE promoter for the production of a heterologous protein.

A proteomic view on genome-based signal peptide predictions.

The availability of complete genome sequences has allowed the prediction of all exported proteins of the corresponding organisms with dedicated algorithms. Even though numerous studies report on genome-based predictions of signal peptides and cell retention signals, they lack a proteomic verification. For example, 180 secretory and 114 lipoprotein signal peptides were predicted recently for the Gram-positive eubacterium Bacillus subtilis. In the present studies, proteomic approaches were used to define the extracellular complement of the B. subtilis secretome. Using different growth conditions and a hyper-secreting mutant, approximately 200 extracellular proteins were visualized by two-dimensional (2D) gel electrophoresis, of which 82 were identified by mass spectrometry. These include 41 proteins that have a potential signal peptide with a type I signal peptidase (SPase) cleavage site, and lack a retention signal. Strikingly, the remaining 41 proteins were predicted previously to be cell associated because of the apparent absence of a signal peptide (22), or the presence of specific cell retention signals in addition to an export signal (19). To test the importance of the five type I SPases and the unique lipoprotein-specific SPase of B. subtilis, the extracellular proteome of (multiple) SPase mutants was analyzed. Surprisingly, only the processing of the polytopic membrane protein YfnI was strongly inhibited in Spase I mutants, showing for the first time that a native eubacterial membrane protein is a genuine Spase I substrate. Furthermore, a mutation affecting lipoprotein modification and processing resulted in the shedding of at least 23 (lipo-)proteins into the medium. In conclusion, our observations show that genome-based predictions reflect the actual composition of the extracellular proteome for approximately 50%. Major problems are currently encountered with the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins.

TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway.

The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains two tatC-like genes denoted tatCd and tatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which approximately 6-14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present in B. subtilis.

Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome.

One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion. In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.

Conserved serine and histidine residues are critical for activity of the ER-type signal peptidase SipW of Bacillus subtilis.

Type I signal peptidases (SPases) are required for the removal of signal peptides from translocated proteins and, subsequently, release of the mature protein from the trans side of the membrane. Interestingly, prokaryotic (P-type) and endoplasmic reticular (ER-type) SPases are functionally equivalent, but structurally quite different, forming two distinct SPase families that share only few conserved residues. P-type SPases were, so far, exclusively identified in eubacteria and organelles, whereas ER-type SPases were found in the three kingdoms of life. Strikingly, the presence of ER-type SPases appears to be limited to sporulating Gram-positive eubacteria. The present studies were aimed at the identification of potential active site residues of the ER-type SPase SipW of Bacillus subtilis, which is required for processing of the spore-associated protein TasA. Conserved serine, histidine, and aspartic acid residues are critical for SipW activity, suggesting that the ER-type SPases employ a Ser-His-Asp catalytic triad or, alternatively, a Ser-His catalytic dyad. In contrast, the P-type SPases employ a Ser-Lys catalytic dyad (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). Notably, catalytic activity of SipW was not only essential for pre-TasA processing, but also for the incorporation of mature TasA into spores.

The potential active site of the lipoprotein-specific (type II) signal peptidase of Bacillus subtilis.

Type II signal peptidases (SPase II) remove signal peptides from lipid-modified preproteins of eubacteria. As the catalytic mechanism employed by type II SPases was not known, the present studies were aimed at the identification of their potential active site residues. Comparison of the deduced amino acid sequences of 19 known type II SPases revealed the presence of five conserved domains. The importance of the 15 best conserved residues in these domains was investigated using the type II SPase of Bacillus subtilis, which, unlike SPase II of Escherichia coli, is not essential for viability. The results showed that only six residues are important for SPase II activity. These are Asp-14, Asn-99, Asp-102, Asn-126, Ala-128, and Asp-129. Only Asp-14 was required for stability of SPase II, indicating that the other five residues are required for catalysis, the active site geometry, or the specific recognition of lipid-modified preproteins. As Asp-102 and Asp-129 are the only residues invoked in the known catalytic mechanisms of proteases, we hypothesize that these two residues are directly involved in SPase II-mediated catalysis. This implies that type II SPases belong to a novel family of aspartic proteases.

Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis.

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis alpha-amylase (AmyL), Escherichia coli TEM beta-lactamase (Bla), human pancreatic alpha-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.

Different mechanisms for thermal inactivation of Bacillus subtilis signal peptidase mutants.

The type I signal peptidase SipS of Bacillus subtilis is of major importance for the processing of secretory precursor proteins. In the present studies, we have investigated possible mechanisms of thermal inactivation of five temperature-sensitive SipS mutants. The results demonstrate that two of these mutants, L74A and Y81A, are structurally stable but strongly impaired in catalytic activity at 48 degrees C, showing the (unprecedented) involvement of the conserved leucine 74 and tyrosine 81 residues in the catalytic reaction of type I signal peptidases. This conclusion is supported by the crystal structure of the homologous signal peptidase of Escherichia coli (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). In contrast, the SipS mutant proteins R84A, R84H, and D146A were inactivated by proteolytic degradation, indicating that the conserved arginine 84 and aspartic acid 146 residues are required to obtain a protease-resistant conformation. The cell wall-bound protease WprA was shown to be involved in the degradation of SipS D146A, which is in accord with the fact that SipS has a large extracytoplasmic domain. As WprA was not involved in the degradation of the SipS mutant proteins R84A and R84H, we conclude that multiple proteases are responsible for the thermal inactivation of temperature-sensitive SipS mutants.

The plasmid-encoded signal peptidase SipP can functionally replace the major signal peptidases SipS and SipT of Bacillus subtilis.

The gram-positive eubacterium Bacillus subtilis is the organism with the largest number of paralogous type I signal peptidases (SPases) known. These are specified both by chromosomal and plasmid-borne genes. The chromosomally encoded SPases SipS and SipT have a major function in precursor processing, and cells depleted of SipS and SipT stop growing and die. In this study, we show that the SPase SipP, specified by the B. subtilis plasmid pTA1015, can functionally replace SipS and SipT, unlike the three chromosomally encoded SPases with a minor function in protein secretion (i.e., SipU, SipV, and SipW). Unexpectedly, SipP is not specifically required for the processing and secretion of Orf1p, which is specified by a gene that is cotranscribed with sipP. These two genes form a conserved structural module of rolling-circle plasmids from B. subtilis. As previously shown for the chromosomal sipS and sipT genes, the transcription of plasmid-borne copies of sipP is temporally controlled, reaching maximal levels during the post-exponential growth phase when the cells secrete proteins at high levels. However, increased transcription of sipP starts at the end of exponential growth, about 2 h earlier than that of sipS and sipT. These data suggest that SipP fulfills a general role in the secretory precursor processing of pTA1015-containing cells.

The role of lipoprotein processing by signal peptidase II in the Gram-positive eubacterium bacillus subtilis. Signal peptidase II is required for the efficient secretion of alpha-amylase, a non-lipoprotein.

Computer-assisted analyses indicate that Bacillus subtilis contains approximately 300 genes for exported proteins with an amino-terminal signal peptide. About 114 of these are lipoproteins, which are retained in the cytoplasmic membrane. We have investigated the importance of lipoprotein processing by signal peptidase II (SPase II) for cellular homeostasis, using cells lacking SPase II. The results show that lipoprotein processing is important for cell viability at low and high temperatures, suggesting that lipoproteins are essential for growth under these conditions. Although certain lipoproteins are required for the development of genetic competence, sporulation, and germination, these developmental processes were not affected in the absence of SPase II. Cells lacking SPase II accumulated lipid-modified precursor and mature-like forms of PrsA, a folding catalyst for secreted proteins. These forms of PrsA seem to have a reduced activity, as the secretion of alpha-amylase was strongly impaired. Unexpectedly, type I signal peptidases, which process secretory preproteins, were not involved in alternative amino-terminal processing of pre-PrsA in the absence of SPase II. In conclusion, processing of lipoproteins by SPase II in B. subtilis is not strictly required for lipoprotein function, which is surprising as lipoproteins and type II SPases seem to be conserved in all eubacteria.

Subunit II of Bacillus subtilis cytochrome c oxidase is a lipoprotein.

The sequence of the N-terminal end of the deduced ctaC gene product of Bacillus species has the features of a bacterial lipoprotein. CtaC is the subunit II of cytochrome caa3, which is a cytochrome c oxidase. Using Bacillus subtilis mutants blocked in lipoprotein synthesis, we show that CtaC is a lipoprotein and that synthesis of the membrane-bound protein and covalent binding of heme to the cytochrome c domain is not dependent on processing at the N-terminal part of the protein. Mutants blocked in prolipoprotein diacylglyceryl transferase (Lgt) or signal peptidase type II (Lsp) are, however, deficient in cytochrome caa3 enzyme activity. Removal of the signal peptide from the CtaC polypeptide, but not lipid modification, is seemingly required for formation of functional enzyme.

Protein secretion and possible roles for multiple signal peptidases for precursor processing in bacilli.

Bacillus subtilis is one of the best known Gram-positive bacteria at both the genetic and physiological level. The entire sequence of its chromosome is known and efficient tools for the genetic modification of this bacterium are available. Moreover, B. subtilis and related Bacillus species are widely used in biotechnology, in particular for the production of secreted enzymes. Although bacilli can secrete large amounts of several native enzymes, the use of these bacteria for the production of heterologous enzymes has frequently resulted in low yields. Here we describe the identification of several components of the Bacillus protein secretion machinery. These components can now be engineered for optimal protein secretion. Special emphasis is given on type I signal peptidases, which remove signal peptides from secretory precursor proteins. Five genes specifying such enzymes (sip, for signal peptidase) are present on the B. subtilis chromosome. Although none of the sip genes is essential by itself, a specific combination of mutations in these genes is lethal. The expression pattern of some of the sip genes coincides with that of many secretory proteins, which seems to reflect an adaptation to high demands on the secretion machinery. Although the various B. subtilis type I signal peptidases have at least partially overlapping substrate specificities, clear differences in substrate preferences are also evident. These observations have implications for the engineering of the processing apparatus for improved secretion of native and heterologous proteins by Bacillus.

Functional analysis of the secretory precursor processing machinery of Bacillus subtilis: identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases.

Approximately 47% of the genes of the Gram-positive bacterium Bacillus subtilis belong to paralogous gene families. The present studies were aimed at the functional analysis of the sip gene family of B. subtilis, consisting of five chromosomal genes, denoted sipS, sipT, sipU, sipV, and sipW. All five sip genes specify type I signal peptidases (SPases), which are actively involved in the processing of secretory preproteins. Interestingly, strains lacking as many as four of these SPases could be obtained. As shown with a temperature-sensitive SipS variant, only cells lacking both SipS and SipT were not viable, which may be caused by jamming of the secretion machinery with secretory preproteins. Thus, SipS and SipT are of major importance for protein secretion. This conclusion is underscored by the observation that only the transcription of the sipS and sipT genes is temporally controlled via the DegS-DegU regulatory system, in concert with the transcription of most genes for secretory preproteins. Notably, the newly identified SPase SipW is highly similar to SPases from archaea and the ER membrane of eukaryotes, suggesting that these enzymes form a subfamily of the type I SPases, which is conserved in the three domains of life.

The Sip(Sli) gene of Streptomyces lividans TK24 specifies an unusual signal peptidase with a putative C-terminal transmembrane anchor.

Type I signal peptidases (SPases) are a widespread family of enzymes which remove signal peptides from proteins translocated across cellular membranes. Here, we report the first isolation of a gene coding for type I signal peptidase of Streptomyces, denoted Sip(Sli). The sip(sli) gene specifies a protein of 291 amino acids. Thus Sip(Sli) is much larger (approximately 100 amino acids) than other known SPases of Gram-positive bacteria and resembles SPases of Gram-negative bacteria, showing the highest degree of similarity to an SPase of the cyanobacterium Phormidium laminosum. Sip(Sli) contains conserved serine and lysine residues, which are believed to be required for the catalytic activity. Similar to other known SPases from Gram-positive bacteria, Sip(Sli) seems to have only one N-terminal transmembrane anchor. In addition, Sip(Sli) seems to contain a second transmembrane anchor at the C-terminus, which is an unusual feature for type I signal peptidases.

Bacillus subtilis contains four closely related type I signal peptidases with overlapping substrate specificities. Constitutive and temporally controlled expression of different sip genes.

Most biological membranes contain one or two type I signal peptidases for the removal of signal peptides from secretory precursor proteins. In this respect, the Gram-positive bacterium Bacillus subtilis seems to be exceptional, because it contains at least four chromosomally-encoded type I signal peptidases, denoted SipS, SipT, SipU, and SipV. Here, we report the identification of the sipT and sipV genes, and the functional characterization of SipT, SipU, and SipV. The four signal peptidases have similar substrate specificities, as they can all process the same beta-lactamase precursor. Nevertheless, they seem to prefer different pre-proteins, as indicated by studies on the processing of the pre-alpha-amylase of Bacillus amyloliquefaciens in strains lacking SipS, SipT, SipU, or SipV. The sipU and sipV genes are constitutively transcribed at a low level, suggesting that they are required for processing of (pre-)proteins secreted during all growth phases. In contrast, the transcription of sipS and sipT is temporally controlled, in concert with the expression of the genes for most secretory proteins, which suggests that SipS and SipT serve to increase the secretory capacity of B. subtilis. Taken together, our findings suggest that SipS, SipT, SipU, and SipV serve different functions during the exponential and post-exponential growth phase of B. subtilis.