Pitt-Hopkins syndrome (PHS) is a rare syndromic mental disorder, which is mainly characterized by severe motor and mental retardation including absent language development, a characteristic facial gestalt and episodes of hyperventilation. We report on a female patient with PHS showing severe mental retardation with absent speech, pronounced muscular hypotonia, ataxia, distinctive facial features, such as a coarse face, a broad nasal bridge and a wide mouth, and hyperventilation attacks. In this patient, genomic profiling by array-based comparative genomic hybridization and fluorescence in situ hybridization studies detected and confirmed a de novo 0.5 Mb deletion in 18q21.2 containing a single gene, the basic helix-loop-helix transcription factor TCF4. cDNA and genomic analyses in the patient and her parents demonstrated TCF4 haploinsufficiency as the underlying cause of the disease. Analysis of the embryonal expression pattern of the Danio rerio ortholog, tcf4, by whole-mount in situ hybridization showed a highly specific expression domain in the pallium of the telencephalon during late somitogenesis, when the patterning of the zebrafish brain is advanced and neural differentiation commences. Later expression domains were restricted to several regions in the central nervous system, including continued expression in the pallium of the telencephalon, and starting expression in the diencephalon (thalamus, ventral thalamus and posterior tuberculum), the midbrain tegmentum, the hindbrain and the branchial arches. This expression pattern correlates with the clinical phenotype. Our results show that haploinsufficiency of TCF4 causes PHS and suggest that D. rerio is a valuable model to study the molecular pathogenesis of PHS and the role of TCF4 in brain development.

Staphylococcus aureus expresses various cell wall-associated and extracellular virulence factors, coordinately controlled by different two-component signal transduction systems and transcriptional regulators. In this study, we used microarray technology to identify the genes regulated by ArlR. The microarray data indicate that ArlR functions as a positive regulator and also as a negative repressor to directly and/or indirectly mediate the expression of at least 114 genes involved in different functions, including autolysis, cell division, growth, and pathogenesis.Staphylococcus aureus is an important human and animal pathogen that causes a wide range of infections, including life-threatening endocarditis and toxic shock syndrome (22,26). The ability of this organism to cause a variety of diseases is partly due to the expression of different cell wall-associated and secreted virulence factors which enable the bacteria to adhere to and colonize host cells (13) or cause toxic shock syndrome (26). The expression of virulence factors is coordinately controlled by two-component signal transduction systems, such as agr (1), sae (16), arl (14), and srrAB (30), and global regulators, including sar (8, 9), sigB (3, 31), rot (2, 24), and mgr (23). Therefore, the elucidation of the regulons of these regulatory systems is important for better understanding molecular mechanisms of pathogenesis. Recently, S. aureus regulons of agr, sar, sigB, and rot have been revealed by using a microarray-based approach (4,11,27). In our studies, we identified target genes controlled by ArlR by a comparison of the transcriptional profile between an arlR mutant and the wild-type strain during the mid-exponential phase of growth by using Affymetrix S. aureus arrays.Construction of the arlR deletion mutant. The arlR deletion mutant (Sa316ko) was constructed by bacteriophage 11-mediated transduction of a cassette containing the tetA gene, flanked by chromosomal fragments upstream and downstream of the alrR, from strain RN4220 into a clinical human isolate strain WCUH29 as described previously (12). Selection for tetracycline resistance and screening for the loss of the erythromycin resistance marker carried by the vector indicated that allelic replacement had occurred and resulted in the arlR mutant strain Sa316ko. The mutation in arlR was verified by PCR and Southern blot analysis (data not shown).To characterize the arlR mutant strain, we examined the effect of the arlR mutation on the bacterial growth rate, CFU, phenotype, and stress responses to different antibacterial agents, including cell wall synthesis inhibitors such as bacitracin, phosphomycin, and vancomycin. No significant effect of the arlR mutation on stress response to antibacterial agents was observed (data not shown). However, Sa316ko grew slightly slower than WCUH29 between early log and stationary phases (Fig. 1). Growth curves as measured by optical density (OD) were confirmed by determining the effect of arlR mutation on colony size and viable cell counts at intervals during...

Staphylococcus aureus is a major human and animal pathogen. During infection, this organism not only is able to attach to and enter host cells by using its cell surface-associated factors but also exports toxins to induce apoptosis and kill invaded cells. In this study, we identified the regulon of a two-component signal transduction system, SaeRS, and demonstrated that the SaeRS system is required for S. aureus to cause infection both in vitro and in vivo. Using microarray and real-time reverse transcriptase PCR analyses, we found that SaeRS regulates the expression of genes involved in adhesion and invasion (such as those encoding fibronectin-binding proteins and fibrinogen-binding proteins) and genes encoding ␣-, ␤-, and ␥-hemolysins. Surprisingly, we found that SaeRS represses the Agr regulatory system since the mutation of saeS up-regulates agrA expression, which was confirmed by using an agr promoter-reporter fusion system. More importantly, we demonstrated that inactivation of the SaeRS system significantly decreases the bacterium-induced apoptosis and/or death of lung epithelial cells (A549) and attenuates virulence in a murine infection model. Moreover, we found that inactivation of the SaeRS system eliminates staphylococcal adhesion and internalization of lung epithelial cells. We also found that both a novel hypothetical protein (the SA1000 protein) and a bifunctional protein (Efb), which binds to extracellular fibrinogen and complement factor C3, might partially contribute to bacterial adhesion to and invasion of epithelial cells. Our results indicate that activation of the SaeRS system may be required for S. aureus to adhere to and invade epithelial cells.

We could identify novel microimbalances as the probable cause of mental retardation in 10% of patients with unclear etiology. The gene content of the microimbalances was found to correlate with phenotype severity. Precise breakpoint analyses allowed the identification of deleted genes presumably causing mental retardation.

Two-component signal transduction systems play an important role in the ability of bacteria to adapt to various environments by sensing changes in their habitat and by altering gene expression. In this study, we report a novel two-component system, YhcSR, in Staphylococcus aureus which is required for bacterial growth in vitro. We found that the down-regulation of yhcSR expression by induced yhcS antisense RNA can inhibit and terminate bacterial growth. Moreover, without complementary yhcS or yhcR, no viable yhcS or yhcR gene replacement mutant was recoverable. Collectively, these results demonstrated that the YhcSR regulatory system is indispensable for S. aureus growth in culture. Moreover, induced yhcS antisense RNA selectively increased bacterial susceptibility to phosphomycin. These data suggest that YhcSR probably modulates the expression of genes critical for bacterial survival and may be a potential target for the development of novel antibacterial agents.Staphylococcus aureus is an important community-and hospital-acquired pathogen that causes superficial skin and lifethreatening infections worldwide (25,33). The pathogenesis of S. aureus is partially due to the coordinated regulation of virulence factors allowing the bacterium to evade the host immune system and/or to promote survival during infection. This organism has developed a series of two-component signal transduction systems (TCSs) in order to sense its immediate surroundings and to modulate specific cellular responses and the expression of virulence genes (14, 32). Therefore, TCSs are being explored as potential targets for new antimicrobials (2,17,28).A typical TCS is composed of a membrane-associated histidine kinase, which acts as a sensor protein extending through the cytoplasmic membrane to monitor environmental changes and to activate a response regulator existing in the cytoplasm modulating gene expression (15,34). The well-studied TCS Agr is a positive regulator of exoproteins, including proteases, hemolysins, and toxins (32). Additionally, the TCS Agr is a repressor of the transcription of protein A, coagulase, and some adhesins in late-exponential-phase growth in vitro (32). Other two-component systems, such as saeRS, arlRS, and srrAB, also influence the expression of some virulence factors (1,13,11,35,39). Another system, LytSR, controls bacterial autolysis by positively regulating the transcription of genes responsible for the synthesis and transport of cell wall murein hydrolase (4). The vraSR loci are homologous to the yvqEC loci of B. subtilis and positively modulate cell wall biosynthesis in S. aureus (23).The yycFG system, which has orthologs in Bacillus subtilis (7,8) and Streptococcus pneumoniae (36), is the only known regulatory system essential for cell viability in S. aureus (26). It was reported that in B. subtilis the YycFG system controls the ftsAZ operon, which is involved in the process of cell wall division (12). However, there is no such evidence in S. aureus and S. pneumoniae, while it has been reported that YycF ...