Summary
The NAD+ and NADH‐sensing transcriptional regulator Rex is widely conserved across gram‐positive bacteria. Rex monitors cellular redox poise and controls the expression of genes/operons ...involved in diverse pathways including alternative fermentation, oxidative stress responses, and biofilm formation. The oral cavity undergoes frequent and drastic fluctuations in nutrient availability, pH, temperature, oxygen tension, saliva, and shear forces. The oral streptococci are major colonizers of oral mucosa and tooth surfaces and include commensals as well as opportunistic pathogens, including the primary etiological agent of dental caries, Streptococcus mutans. Current understanding of the Rex regulon in oral bacteria is mostly based on studies in S. mutans and endodontic pathogen Enterococcus faecalis. Indeed, other oral bacteria encode homologs of the Rex protein and much is to be gleaned from more in‐depth studies. Our current understanding has Rex positioned at the interface of oxygen and energy metabolism. In biofilms, heterogeneous oxygen tension influences the ratio of intracellular NADH and NAD+, which is finely tuned through glycolysis and fermentation. In S. mutans, Rex regulates the expression of glycolytic enzyme NAD+‐dependent glyceraldehyde 3‐phosphate dehydrogenase, and NADH‐dependent fermentation enzymes/complexes lactate dehydrogenase, pyruvate dehydrogenase, alcohol‐acetaldehyde dehydrogenase, and fumarate reductase. In addition, Rex controls the expression of NADH oxidase, a major enzyme used to eliminate oxidative stress and regenerate NAD+. Here, we summarize recent studies carried out on the Rex regulators in S. mutans and E. faecalis. This research has important implications for understanding how Rex monitors redox balance and optimizes fermentation pathways for survival and subsequent pathogenicity.
Summary
Our previous studies showed that brpA in Streptococcus mutans, which encodes a member of the LytR‐CpsA‐Psr family of proteins, can be co‐transcribed with brpB upstream as a bicistronic ...operon, and the intergenic region also has strong promoter activity. To elucidate how brpA expression is regulated, the promoter regions were analyzed using polymerase chain reaction‐based deletions and site‐directed mutagenesis and a promoterless luciferase gene as a reporter. Allelic exchange mutagenesis was also used to examine genes encoding putative trans‐acting factors, and the impact of such mutations on brpA expression was analyzed by reporter assays. Multiple elements in the short brpA promoter (nucleotide −1 to −344 relative to start cordon ATG) were shown to have a major impact on brpA expression, including an FNR‐box, for a putative binding site of an FNR‐type of transcriptional regulator. When compared with the intact brpA promoter, mutations of the highly conserved nucleotides in FNR‐box from TTGATgtttAcCtt to TTACAgaaaGtTac resulted in 1362‐fold increases of luciferase activity (P < .001), indicative of the FNR‐box‐mediated repression as a major mechanism in regulation of brpA expression. When luciferase reporter was fused to the upstream brpBA promoter (nucleotides −784 to −1144), luciferase activity was decreased by 4.5‐fold (P < .001) in the brpA mutant, TW14D, and by 67.7‐fold (P < .001) in the brpB mutant, JB409, compared with the wild‐type, UA159. However, no such effects were observed when the reporter gene was fused to the short brpA promoter and its derivatives. These results also suggest that brpA expression in S. mutans is auto‐regulated through the upstream brpBA promoter.
NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD(+). The critical nature ...of Nox is 2-fold: it serves to regenerate NAD(+), a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD(+) have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD(+) affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress.
Summary
Streptococcus mutans, a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The SMU864 locus, designated pdxR, is predicted to encode ...a member of the novel MocR/GabR family proteins, which are featured with a winged helix DNA‐binding N‐terminal domain and a C‐terminal domain highly homologous to the pyridoxal phosphate‐dependent aspartate aminotransferases. A pdxR‐deficient mutant, TW296, was constructed using allelic exchange. PdxR deficiency in S. mutans had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain, UA159, the PdxR‐deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (P < 0.01). When analyzed by real‐time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (SMU865) and a pyridoxal permease (SMU866) of the salvage pathway of vitamin B6 biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR‐deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B6. Further studies revealed that although S. mutans is known to require vitamin B6 to grow in defined medium, B6 vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against S. mutans growth and biofilm formation. Our results suggest that PdxR in S. mutans plays an important role in regulation of vitamin B6 metabolism, acid tolerance response and biofilm formation.
Summary
Streptococcus mutans,
a key etiological agent of the human dental caries, lives primarily on the tooth surface in tenacious biofilms. The
SMU
864 locus, designated
pdxR,
is predicted to ...encode a member of the novel MocR/GabR family proteins, which are featured with a winged helix
DNA
‐binding N‐terminal domain and a C‐terminal domain highly homologous to the pyridoxal phosphate‐dependent aspartate aminotransferases. A
pdxR
‐deficient mutant,
TW
296, was constructed using allelic exchange. PdxR deficiency in
S. mutans
had little effect on cell morphology and growth when grown in brain heart infusion. However, when compared with its parent strain,
UA
159, the PdxR
‐
deficient mutant displayed major defects in acid tolerance response and formed significantly fewer biofilms (
P
< 0.01). When analyzed by real‐time polymerase chain reaction, PdxR deficiency was found to drastically reduce expression of an apparent operon encoding a pyridoxal kinase (
SMU
865) and a pyridoxal permease (
SMU
866) of the salvage pathway of vitamin B
6
biosynthesis. In addition, PdxR deficiency also altered the expression of genes for ClpL protease, glucosyltransferase B and adhesin SpaP, which are known to play important roles in stress tolerance and biofilm formation. Consistently, PdxR‐deficiency affected the growth of the deficient mutant when grown in defined medium with and without vitamin B
6
. Further studies revealed that although
S. mutans
is known to require vitamin B
6
to grow in defined medium, B
6
vitamers, especially pyridoxal, were strongly inhibitory at millimolar concentrations, against
S. mutans
growth and biofilm formation. Our results suggest that PdxR in
S. mutans
plays an important role in regulation of vitamin B
6
metabolism, acid tolerance response and biofilm formation.
Purpose
Vibration Tonometry enables precise non‐contact tonometry independently of CCT and post LASIK using induced corneal vibrations response to calculate IOP using different algorithms for men and ...women. This corneal vibrations response reflects different tissue elasticity between men and women. We calculate IOP error using gender‐inverted algorithms: female algorithm for men and vice versa
Methods
Vibration Tonometry IOP data from clinical trials in 324 subjects in 3 IOP groups and in 3 age groups. 1,040 and 1,031 measurements were used respectively in women and in men
Results
Using men algorithm for women mean IOP difference was −0.376 mm Hg; 0.07 mmHg; 1.937 mmHg respectively in the <16 mm; 16–23 mm and in the >23 mm Hg group significantly different p < 0.0001. In 3 age groups women showed IOP mean difference of −0.086 mm Hg, 0.077 and −0.224 mm Hg respectively in <50; 50–60 and >60 years old women. Kruskal Wallis test used showed all 3 groups differed significantly p < 0.001, when compared 2 to 2 the error in the 50–60 group differed significantly from that in >60 years old group. For Men mean IOP difference was −0.115 mm Hg in the <16 mm group versus 0.385 in the 16–26 mm Hg group and −1.002 in the >23 mm Hg group all significantly different (Ttest p < 0.0009). The 3 age groups in men showed IOP mean difference of −0.129 mmHg, 0.185 and 0.259 mmHg (SD = 1.286) respectively in <50; 50–60 and >60 years old men. Kruskal Wallis test non significant p < 0.056, when compared 2 to 2 the error in the <50 group differed nearly significantly from that in >60 years old p = 0.025
Conclusions
Gender related difference in IOP measurement is most significant in the higher IOP, >60 years old and most significantly in women with higher corneal elasticity. These analyses confirm the important need for gender specific tonometry as uniquely provided by Vibration Tonometry.
Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a putative serine protease inhibitor encoded by serine protease inhibitor Kazal-type 5 (SPINK5). It is strongly expressed in differentiated ...keratinocytes in normal skin but expression is markedly reduced or absent in Netherton syndrome (NS), a severe ichthyosis caused by SPINK5 mutations. At present, however, both the precise intracellular localization and biological roles of LEKTI are not known. To understand the functional role of LEKTI, we examined the localization of LEKTI together with kallikrein (KLK)7 and KLK5, possible targets of LEKTI, in the human epidermis, by confocal laser scanning microscopy and immunoelectron microscopy. In normal skin, LEKTI, KLK7, and KLK5 were all found in the lamellar granule (LG) system, but were separately localized. LEKTI was expressed earlier than KLK7 and KLK5. In NS skin, LEKTI was absent and an abnormal split in the superficial stratum granulosum was seen in three of four cases. Collectively, these results suggest that in normal skin the LG system transports and secretes LEKTI earlier than KLK7 and KLK5 preventing premature loss of stratum corneum integrity/cohesion. Our data provide new insights into the biological functions of LG and the pathogenesis of NS.
Purpose
Novel non‐contact, non‐invasive Vibration Tonometry using analysis of induced corneal vibrations and their correlation to GAT IOP was tested in a large multicenter clinical trial.
Methods
470 ...eyes in 236 volunteers from various ethnic origins at Paris area hospital eye clinic and Glaucoma Institute were studied. History, vibration tonometry measurements in triplicate GAT, NIDEK air puff IOP, refractometry, keratometry and non‐contact pachymetry. Algorithm discovery was performed by random draw splitting runs into a discovery sample and a separate validation sample performed 10 times separately in men and women
Results
In 236 patients, 34.6% of eyes had glaucoma, 51% of which women. 1311/1772 (76%) vibration tonometry measurements were in correct position. Population aged 20–92 yo with 48.7% females with mean of 62.1(± 13.5) in women and 62.5 (± 12.7) in men. CCT mean was 531.2 μ (± 37.3) in women and 534.8 μ (± 38.5) in men. 207 patients AL mean was 23.29 mm (± 1.68) in women and 23.73 mm (± 1.68) in men. GAT IOP ranged from 5.3 to 34 mm Hg, mean was 15.2 mm Hg (± 3.1) in women and 15.2 (± 3.52) in men ‐No significant differences. No adverse events during or post trial. Algorithm for women yielded 98.6% of IOP mean within 5 mm Hg of mean GAT IOP and 86% within 3 mm Hg. For men 96.4% of IOP mean was within 5 mm Hg of mean GAT IOP and 84.2% within 3 mm Hg from GAT IOP mean. Using the mean of 2 closest measures was used .
Conclusions
Current algorithm shows no influence of CCT on IOP. Gender Specific algorithm concurrs with previous publications of different corneal hysteresis (lower) in women. Vibration tonometry is a valuable method for measurement of IOP without contact with precision and reproducibility independently of CCT. It may be a valuable tool for IOP measurement in thin and surgical corneas.
Background: Smith-Magenis syndrome (SMS) is rare (prevalence 1 in 25 000) and is associated with psychomotor delay, a particular behavioural pattern and congenital anomalies. SMS is often due to a ...chromosomal deletion of <4 Mb at the 17p11.2 locus, leading to haploinsufficiency of numerous genes. Mutations of one of these gemes, RAI1, seems to be responsible for the main features found with heterozygous 17p11.2 deletions. Methods: We studied DNA from 30 patients with SMS using a 300 bp amplimers comparative genome hybridisation array encompassing 75 loci from a 22 Mb section from the short arm of chromosome 17. Results: Three patients had large deletions (10%). Genotype–phenotype correlation showed that two of them had cleft palate, which was not found in any of the other patients with SMS (p<0.007, Fisher’s exact test). The smallest extra-deleted region associated with cleft palate in SMS is 1.4 Mb, contains <16 genes and is located at 17p11.2-17p12. Gene expression array data showed that the ubiquitin B precursor (UBB) is significantly expressed in the first branchial arch in the fourth and fifth weeks of human development. Conclusion: These data support UBB as a good candidate gene for isolated cleft palate.
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