WhyD tailors surface polymers to prevent bacteriolysis and direct cell elongation in Streptococcus pneumoniae

Penicillin and related antibiotics disrupt cell wall synthesis in bacteria and induce lysis by misactivating cell wall hydrolases called autolysins. Despite the clinical importance of this phenomenon, little is known about the factors that control autolysins and how penicillins subvert this regulation to kill cells. In the pathogen Streptococcus pneumoniae (Sp), LytA is the major autolysin responsible for penicillin-induced bacteriolysis. We recently discovered that penicillin treatment of Sp causes a dramatic shift in surface polymer biogenesis in which cell wall-anchored teichoic acids (WTAs) increase in abundance at the expense of lipid-linked lipoteichoic acids. Because LytA binds to these polymers, this change recruits the enzyme to its substrate where it cleaves the cell wall and elicits lysis. In this report, we identify WhyD (SPD_0880) as a new factor that controls the level of WTAs in Sp cells to prevent LytA misactivation and lysis. We show that WhyD is a WTA hydrolase that restricts the WTA content of the wall to areas adjacent to active PG synthesis. Our results support a model in which the WTA tailoring activity of WhyD directs PG remodeling activity required for proper cell elongation in addition to preventing autolysis by LytA.

LcpB Is a Pyrophosphatase Responsible for Wall Teichoic Acid Synthesis and Virulence in Staphylococcus aureus Clinical Isolate ST59

The community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes severe pandemics primarily consisting of skin and soft tissue infections. However, the underlying pathomechanisms of the bacterium are yet to fully understood. The present study identifies LcpB protein, which belongs to the LytR-A-Psr (LCP) family, is crucial for cell wall synthesis and virulence in S. aureus. The findings revealed that LcpB is a pyrophosphatase responsible for wall teichoic acid synthesis. The results also showed that LcpB regulates enzyme activity through specific key arginine sites in its LCP domain. Furthermore, knockout of lcpB in the CA-MRSA isolate ST59 resulted in enhanced hemolytic activity, enlarged of abscesses, and increased leukocyte infiltration. Meanwhile, we also found that LcpB regulates virulence in agr-independent manner and the key sites for pyrophosphatase of LcpB play critical roles in regulating the virulence. In addition, the results showed that the role of LcpB was different between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). This study therefore highlights the dual role of LcpB in cell wall synthesis and regulation of virulence. These insights on the underlying molecular mechanisms can thus guide the development of novel anti-infective strategies.

SmdA is a novel cell morphology determinant in Staphylococcus aureus

Cell division and cell wall synthesis in staphylococci need to be precisely coordinated and controlled to allow the cell to multiply while maintaining their nearly spherical shape. The mechanisms ensuring correct placement of the division plane and synthesis of new cell wall have been studied intensively, however, hitherto unknown factors and proteins are likely to play key roles in this complex interplay. Starting from a subcellular localization- and gene knockdown screen of essential genes with unknown functions in Staphylococcus aureus, we identified a protein with major influence on cell morphology in S. aureus. The protein, here named SmdA (for staphylococcal morphology determinant A), is a membrane-protein with septum-enriched localization. By smdA silencing and overexpression, we have used different microscopy techniques to show that SmdA is critical for cell division, including septum formation and cell splitting. We also identified conserved residues in SmdA that are critical for functionality. Pulldown- and bacterial two-hybrid interaction experiments showed that SmdA interacts with several known cell division- and cell wall synthesis proteins, including penicillin binding proteins (PBPs) and EzrA. Notably, SmdA also affects susceptibility to cell wall targeting antibiotics, particularly in methicillin-resistant S. aureus (MRSA). Together, our results show that S. aureus is dependent on balanced amounts of membrane-attached SmdA in order to carry out proper cell division.

Exogenous proanthocyanidins improve tolerance of Cu-toxicity by amelioration of oxidative damage and re-programming of gene expression in Medicago sativa

Excess copper (Cu) in soil due to industrial and agricultural practices can result in reduced plant growth. Excess Cu resulted in severely retarded root growth with severe discoloration of Alfalfa (Medicago sativa) and Medicago truncatula. Growth in the presence of hydrogen peroxide resulted in similar symptoms that could be partially recovered by the addition of the reductant ascorbic acid revealing damage was likely due to oxidative stress. The addition of proanthocyanidins (PAs) in the presence of Cu prevented much of the damage, including plant growth and restoration of lignin synthesis which was inhibited in the presence of excess Cu. Transcriptome analyses of the impact of excess Cu and the amelioration after PAs treatment revealed that changes were enriched in functions associated with the cell wall and extracellular processes, indicating that inhibition of cell wall synthesis was likely the reason for retarded growth. Excess Cu appeared to induce a strong defense response, along with alterations in the expression of a number of genes encoding transcription factors, notably related to ethylene signaling. The addition of PAs greatly reduced this response, and also induced novel genes that likely help ameliorate the effects of excess Cu. These included induction of genes involved in the last step of ascorbic acid biosynthesis and of enzymes involved in cell wall synthesis. Combined, these results show that excess Cu causes severe oxidative stress damage and inhibition of cell wall synthesis, which can be relieved by the addition of PAs.

Analysis of Phenotypic Characteristics and Sucrose Metabolism in the Roots of Raphanus sativus L.

The taproot of radish (Raphanus sativus L.) is an important sink organ; it is morphologically diverse and contains large amounts of secondary metabolites. Sucrose metabolism is believed to be important in the development of sink organs. We measured the amounts of glucose, fructose, and sucrose in the roots of sixty three radish accessions and analyzed the association between the sugar content and the root phenotype. Fructose content correlated with the root color and length characteristics, glucose was the most abundant sugar in the roots, and the sucrose content was very low, compared to that of the hexoses in most of the accessions. Expression analysis of the genes involved in sucrose metabolism, transportation, starch synthesis, and cell wall synthesis was performed through RNA sequencing. The genes encoding sucrose synthases (SUSY) and the enzymes involved in the synthesis of cellulose were highly expressed, indicating that SUSY is involved in cell wall synthesis in radish roots. The positive correlation coefficient (R) between the sucrose content and the expression of cell wall invertase and sugar transporter proteins suggest that hexose accumulation could occur through the apoplastic pathway in radish roots. A positive R score was also obtained when comparing the expression of genes encoding SUSY and fructokinase (FK), suggesting that the fructose produced by SUSY is mostly phosphorylated by FK. In addition, we concluded that sucrose was the most metabolized sugar in radish roots.