Mutation of the NADH oxidase gene (nox) reveals an overlap of the oxygen- and acid-mediated stress responses in Streptococcus mutans.

NADH oxidase (Nox) is a flavin-containing enzyme used by Streptococcus mutans to reduce dissolved oxygen encountered during growth in the oral cavity. In this study, we characterized the role of the NADH oxidase in the oxidative and acid stress responses of S. mutans. A nox-defective mutant strain of S. mutans and its parental strain, the genomic type strain UA159, were exposed to various oxygen concentrations at pH values of 5 and 7 to better understand the adaptive mechanisms used by the organism to withstand environmental pressures. With the loss of nox, the activities of oxygen stress response enzymes such as superoxide dismutase and glutathione oxidoreductase were elevated compared to those in controls, resulting in a greater adaptation to oxygen stress. In contrast, the loss of nox led to a decreased ability to grow in a low-pH environment despite an increased resistance to severe acid challenge. Analysis of the membrane fatty acid composition revealed that for both the nox mutant and UA159 parent strain, growth in an oxygen-rich environment resulted in high proportions of unsaturated membrane fatty acids, independent of external pH. The data indicate that S. mutans membrane fatty acid composition is responsive to oxidative stress, as well as changes in environmental pH, as previously reported (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). The heightened ability of the nox strain to survive acidic and oxidative environmental stress suggests a multifaceted response system that is partially dependent on oxygen metabolites.

Two Spx proteins modulate stress tolerance, survival, and virulence in Streptococcus mutans.

Previous work suggested that the underlying mechanisms by which the Streptococcus mutans ClpXP protease affects virulence traits are associated with accumulation of two orthologues of the Spx regulator, named SpxA and SpxB. Here, a thorough characterization of strains lacking the spx genes (Delta spxA, Delta spxB, and Delta spxA Delta spxB) revealed that Spx, indeed, participates in the regulation of processes associated with S. mutans pathogenesis. The Delta spxA strain displayed impaired ability to grow under acidic and oxidative stress conditions and had diminished long-term viability at low pH. Although the Delta spxB strain did not show any inherent stress-sensitive phenotype, the phenotypes observed in Delta spxA were more pronounced in the Delta spxA Delta spxB double mutant. By using two in vivo models, we demonstrate for the first time that Spx is required for virulence in a gram-positive pathogen. Microarrays confirmed the global regulatory role of SpxA and SpxB. In particular, SpxA was shown to positively regulate genes associated with oxidative stress, a finding supported by enzymatic assays. SpxB had a secondary role in regulation of oxidative stress genes but appeared to play a larger role in controlling processes associated with cell wall homeostasis. Given the high degree of conservation between Spx proteins of low-GC gram-positive bacteria, these results are likely to have broad implications.

Cochlear developmental defect and background-dependent hearing thresholds in the Jackson circler (jc) mutant mouse.

Jackson circler (jc) is a spontaneous, recessive mouse mutation that results in circling behavior and an impaired acoustic startle response. In this study, we refined the phenotypic and genetic parameters of the original jc mutation and characterized a new mutant allele, jc(2J). In open-field behavior tests, homozygous jc mutants exhibited abnormal circling and ambulatory behavior that was indistinguishable from that of phenotypically similar mutants with defects in the vestibule of the inner ear. The jc/jc and jc(2J)/jc(2J) mice had stable elevated auditory-evoked brainstem response (ABR) thresholds at the 16kHz stimulus of 88+/-9dB sound pressure levels (SPL) and 43+/-11dB SPL, respectively. Peak latencies and peak time intervals were normal in jc mutants. The jc mice showed no measurable distortion-product otoacoustic emissions (DPOAEs) above the system noise floor. In the mutant cochlea, the apical turn failed to form due to the developmental growth arrest of the cochlear duct at the level of the first turn at gestational day 13.5. In a large intrasubspecific intercross, jc localized to a 0.2cM interval at position 25cM on chromosome 10, which is homologous to the human 6q21 region. On CZECHII/Ei and CAST/Ei backgrounds jc/jc mutant hearing thresholds at the 16kHz stimulus were significantly lower than those observed on the C57BL/6J background, with means of 62+/-22dB SPL and 55+/-18dB SPL, respectively. Genome-wide linkage scans of backcross, intercross, and congenic progeny revealed a complex pattern of genetic and stochastic effects.

Sustained cadherin 23 expression in young and adult cochlea of normal and hearing-impaired mice.

Cadherin 23 encodes a single-pass transmembrane protein with 27 extracellular cadherin-domains and localizes to stereocilia where it functions as an inter-stereocilia link. Cadherin 23-deficient mice show congenital deafness in combination with circling behavior as a result of organizational defects in the stereocilia hair bundle; common inbred mouse strains carrying the hypomorphic Cdh23(753A) allele are highly susceptible to sensorineural hearing loss. Here, we show that an antibody (N1086) directed against the intracellular carboxyterminus reacts specifically with cadherin 23 and detects with high sensitivity the isoform devoid of the peptide encoded by exon 68 (CDH23Delta68). Cochlea, vestibule, eye, brain and testis produce the CDH23Delta68 isoform in abundance and form moieties with different molecular weight due to variations in glycosylation content. In the cochlea, CDH23Delta68 expression is highest at postnatal day 1 (P1) and P7; expression is down regulated through P14 and P21 and persists at a low steady-state level throughout adulthood (P160). Furthermore, CDH23Delta68 expression levels in young and adult cochlea are similar among normal and hearing deficient strains (C3HeB/FeJ, C57BL/6J and BUB/BnJ). Finally, by immunofluorescence using an antibody (Pb240) specific for ectodomain 14, we show that cadherin 23 localizes to stereocilia during hair bundle development in late gestation and early postnatal days. Cadherin 23-specific labeling becomes weaker as the hair bundle matures but faint labeling concentrated near the top of stereocilia is still detectable at P35. No labeling of cochlea stereocilia was observed with N1086. In conclusion, our data describe a cadherin 23-specific antibody with high affinity to the CDH23Delta68 isoform, reveal a dynamic cochlea expression and localization profile and show sustained cadherin 23 levels in adult cochlea of normal and hearing-impaired mice.