The Cryptosporidium signaling kinase CDPK5 plays an important role in male gametogenesis and parasite virulence.

The protozoan parasite Cryptosporidium is a leading cause of diarrhea in young children. The parasite's life cycle involves a coordinated and timely progression from asexual to sexual stages, leading to the formation of the transmissible oocyst. Underlying molecular signaling mechanisms orchestrating sexual development are not known. Here, we describe the function of a signaling kinase in Cryptosporidium male gametogenesis. We reveal the expression of Cryptosporidium parvum calcium-dependent protein kinase 5 (CDPK5) during male gamete development and its important role in the egress of mature gametes. Genetic ablation of this kinase results in viable parasites, indicating that this gene is dispensable for parasite survival. Interestingly, cdpk5 deletion decreases parasite virulence and impacts oocyst shedding in immunocompromised mice. Using phosphoproteomics, we identify possible CDPK5 substrates and biological processes regulated by this kinase. Collectively, these findings illuminate parasite cell biology by revealing a mechanism controlling male gamete production and a potential target to block disease transmission.

Global mapping of the Chlamydia trachomatis conventional secreted effector - host interactome reveals CebN interacts with nucleoporins and Rae1 to impede STAT1 nuclear translocation.

Chlamydia trachomatis (C.t.), the leading cause of bacterial sexually transmitted infections, employs a type III secretion system (T3SS) to translocate two classes of effectors, inclusion membrane proteins and conventional T3SS (cT3SS) effectors, into the host cell to counter host defense mechanisms. Here we employed three assays to directly evaluate secretion during infection, validating secretion for 23 cT3SS effectors. As bioinformatic analyses have been largely unrevealing, we conducted affinity purification-mass spectrometry to identify host targets and gain insights into the functions of these effectors, identifying high confidence interacting partners for 21 cT3SS effectors. We demonstrate that CebN localizes to the nuclear envelope in infected and bystander cells where it interacts with multiple nucleoporins and Rae1, blocking STAT1 nuclear import following IFN-γ stimulation. By building a cT3SS effector-host interactome, we have identified novel pathways that are targeted during bacterial infection and have begun to address how C.t. effectors combat cell autonomous immunity.

Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host.

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek's disease is a devastating herpesviral disease of chickens caused by Marek's disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5' ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems.

The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK).

Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Marek's disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK-particularly its kinase activity-is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection.

Universal Sample Preparation Workflow for Plant Phosphoproteomic Profiling.

Mass spectrometry (MS)-based phosphoproteomics is a powerful tool for investigating cell signaling, yet it remains challenging to study plant phosphoproteomes due to the low yield of cell lysis and high complexity of plant lysate. Here we report a streamlined sample preparation workflow to analyze plant phosphoproteomes in a high-throughput manner. This workflow addresses the problem of low yield in the lysis step and eliminates the interferences of pigments and metabolites in plant lysate. Integrating chemical labeling and high pH reverse phase fractionation with this workflow achieves in-depth phosphoproteomic coverage. Notably, the scalability of this approach is demonstrated by systematically analyzing the effect of long-term cold stress in the perturbation of the tomato phosphoproteome. Identification of more than 30,000 phosphopeptides from tomato leaves and more than 5000 kinase-substrate pairs from Arabidopsis create the largest phosphoproteomic and signaling network resource to date.

Proteome Composition of Bovine Amniotic Membrane and Its Potential Role in Corneal Healing.

Purpose: To investigate the protein profile of bovine amniotic membranes (bAM) and to determine putative associations between protein composition in bAM and known corneal healing pathways. Methods: The bAM were acquired from normal full-term births (n = 10), processed, and stored at -80°C for two days. Subsequently, the frozen membranes were thawed at room temperature and prepared for proteomic exploration using high-resolution liquid chromatography-mass spectrometry, followed by bioinformatics analysis. Recently identified corneal healing pathways were contrasted with protein profiles and pathways present in bAM. Results: The analyses identified 2105 proteins, with an interactive network of 1271 nodes (proteins) and 8757 edges (interactions). The proteins with higher betweenness centrality measurements include microfibril-associated protein 4, HSD3B1, CAPNS1, ATP1B3, CAV1, ANXA2, YARS, and GAPDH. The top four pathways in Kyoto Encyclopedia of Genes and Genomes were ribosome, metabolic pathway, spliceosome, and oxidative phosphorylation. The bAM and cornea shared abundant proteins, genome ontology, and signaling pathways. Conclusions: The high-throughput proteomic profile of the bAM demonstrated that numerous proteins present in the cornea are also present in this fetal membrane. Our findings collectively demonstrate the similarity between bAM and the cornea's protein composition, supporting our hypothesis that bAM can be used to treat corneal diseases.

Substrate Recognition by the Class II Lanthipeptide Synthetase HalM2.

Class II lanthipeptides belong to a diverse group of natural products known as ribosomally synthesized and post-translationally modified peptides (RiPPs). Most RiPP precursor peptides contain an N-terminal recognition sequence known as the leader peptide, which is typically recognized by biosynthetic enzymes that catalyze modifications on the C-terminal core peptide. For class II lanthipeptides, these are carried out by a bifunctional lanthipeptide synthetase (LanM) that catalyzes dehydration and cyclization reactions on peptidic substrates to generate thioether-containing, macrocyclic molecules. Some lanthipeptide synthetases are extraordinarily substrate tolerant, making them promising candidates for biotechnological applications such as combinatorial biosynthesis and cyclic peptide library construction. In this study, we characterized the mode of leader peptide recognition by HalM2, the lanthipeptide synthetase responsible for the production of the antimicrobial peptide haloduracin ß. Using NMR spectroscopic techniques, in vitro binding assays, and enzyme activity assays, we identified substrate residues that are important for binding to HalM2 and for post-translational modification of the peptide substrates. Additionally, we provide evidence of the binding site on the enzyme using binding assays with truncated enzyme variants, hydrogen-deuterium exchange mass spectrometry, and photoaffinity labeling. Understanding the mechanism by which lanthipeptide synthetases recognize their substrate will facilitate their use in biotechnology, as well as further our general understanding of how RiPP enzymes recognize their substrates.

Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.

Protein methyltransferases mediate posttranslational modifications of both histone and nonhistone proteins. Whereas histone methylation is well-known to regulate gene expression, the biological significance of nonhistone methylation is poorly understood. Methyltransferase-like 21c (Mettl21c) is a newly classified nonhistone lysine methyltransferase whose in vivo function has remained elusive. Using a Mettl21cLacZ knockin mouse model, we show here that Mettl21c expression is absent during myogenesis and restricted to mature type I (slow) myofibers in the muscle. Using co-immunoprecipitation, MS, and methylation assays, we demonstrate that Mettl21c trimethylates heat shock protein 8 (Hspa8) at Lys-561 to enhance its stability. As such, Mettl21c knockout reduced Hspa8 trimethylation and protein levels in slow muscles, and Mettl21c overexpression in myoblasts increased Hspa8 trimethylation and protein levels. We further show that Mettl21c-mediated stabilization of Hspa8 enhances its function in chaperone-mediated autophagy, leading to degradation of client proteins such as the transcription factors myocyte enhancer factor 2A (Mef2A) and Mef2D. In contrast, Mettl21c knockout increased Mef2 protein levels in slow muscles. These results identify Hspa8 as a Mettl21c substrate and reveal that nonhistone methylation has a physiological function in protein stabilization.

Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase-like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up-regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin-containing protein (Vcp/p97), a key component of the ubiquitin-proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome-mediated protein degradation.-Wang, C., Zhang, B., Ratliff, A. C., Arrington, J., Chen, J., Xiong, Y., Yue, F., Nie, Y., Hu, K., Jin, W., Tao, W. A., Hrycyna, C. A., Sun, X., Kuang, S. Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.

Effect of Preparation Method on the Protein Profile of Equine Amnion Dressings.

The protein content of amnion is thought to be the primary contributor to its efficacy as a biological dressing for wounds. Protein elution into antibiotic processing media has been reported, but the effect of antiseptic-based processing methods is unknown. Amniotic membranes were collected from eight healthy mares. Samples were collected after removal of gross debris. Tissues were subsequently divided and processed with either 0.05% chlorhexidine or 2% iodine/0.25% acetic acid. After protein extraction and trypsin digestion, the proteins were labeled with 8-plex iTRAQ tags, combined, and analyzed by high-resolution liquid chromatography-mass spectrometry. The MaxQuant-Perseus software suite was used to identify and quantify sample proteins, with functional annotation performed in PANTHER. There were 220 unique proteins identified, of which 144 were found in all individuals and across all conditions, several with a known role in wound healing. Contrary to expectations, processing did not significantly alter the protein content of the amnion tissue. Limitations include the small sample size and single time point. These results suggest that either processing method is acceptable for use in the preparation of equine amnion dressings. The role of expressed proteins in the biological activity of amnion dressings remains to be elucidated.

Identification of the Direct Substrates of the ABL Kinase via Kinase Assay Linked Phosphoproteomics with Multiple Drug Treatments.

Ableson tyrosine kinase (ABL) plays essential roles in cell differentiation, division, adhesion, and stress response. However, fusion of the breakpoint cluster region (BCR) to ABL produces constitutive kinase activity that causes chronic myelogenous leukemia (CML). Small molecule tyrosine kinase inhibitors (TKIs) such as imatinib revolutionized the treatment of CML and other cancers, but acquired resistance to these inhibitors is rising. Thus, careful dissection of ABL signaling pathways is needed to find novel drug targets. Here we present a refined proteomic approach for elucidation of direct kinase substrates called kinase assay linked phosphoproteomics (KALIP). Our strategy integrates in vitro kinase assays at both the peptide and protein levels with quantitative tyrosine phosphoproteomics in response to treatment by multiple TKIs. Utilizing multiple TKIs permits elimination of off-target effects of these drugs, and overlapping the in vivo and in vitro data sets allows us to define a list of the most probable kinase substrates. Applying our approach produced a list of 60 ABL substrates, including novel and known proteins. We demonstrate that spleen tyrosine kinase (SYK) is a novel direct substrate of ABL, and we predict our proteomic strategy may facilitate identification of substrates in other cancers that have disrupted kinase signaling.

Universal Plant Phosphoproteomics Workflow and Its Application to Tomato Signaling in Response to Cold Stress.

Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. To address the high complexity and dynamic range of plant proteomes and phosphoproteomes, we present a universal sample preparation procedure that facilitates plant phosphoproteomic profiling. This advanced workflow significantly improves phosphopeptide identifications, enabling deep insight into plant phosphoproteomes. We then applied the workflow to study the phosphorylation events involved in tomato cold tolerance mechanisms. Phosphoproteomic changes of two tomato species (N135 Green Gage and Atacames) with distinct cold tolerance phenotypes were profiled under cold stress. In total, we identified more than 30,000 unique phosphopeptides from tomato leaves, representing about 5500 phosphoproteins, thereby creating the largest tomato phosphoproteomic resource to date. The data, along with the validation through in vitro kinase reactions, allowed us to identify kinases involved in cold tolerant signaling and discover distinctive kinase-substrate events in two tomato species in response to a cold environment. The activation of SnRK2s and their direct substrates may assist N135 Green Gage tomatoes in surviving long-term cold stress. Taken together, the streamlined approach and the resulting deep phosphoproteomic analyses revealed a global view of tomato cold-induced signaling mechanisms.

Identification of Upstream Kinases by Fluorescence Complementation Mass Spectrometry.

Protein kinases and their substrates comprise extensive signaling networks that regulate many diverse cellular functions. However, methods and techniques to systematically identify kinases directly responsible for specific phosphorylation events have remained elusive. Here we describe a novel proteomic strategy termed fluorescence complementation mass spectrometry (FCMS) to identify kinase-substrate pairs in high throughput. The FCMS strategy employs a specific substrate and a kinase library, both of which are fused with fluorescence complemented protein fragments. Transient and weak kinase-substrate interactions in living cells are stabilized by the association of fluorescence protein fragments. These kinase-substrate pairs are then isolated with high specificity and are identified and quantified by LC-MS. FCMS was applied to the identification of both known and novel kinases of the transcription factor, cAMP response element-binding protein (CREB). Novel CREB kinases were validated by in vitro kinase assays, and the phosphorylation sites were unambiguously located. These results uncovered possible new roles for CREB in multiple important signaling pathways and demonstrated the great potential of this new proteomic strategy.

Recent advances in phosphoproteomics and application to neurological diseases.

Phosphorylation has an incredible impact on the biological behavior of proteins, altering everything from intrinsic activity to cellular localization and complex formation. It is no surprise then that this post-translational modification has been the subject of intense study and that, with the advent of faster, more accurate instrumentation, the number of large-scale mass spectrometry-based phosphoproteomic studies has swelled over the past decade. Recent developments in sample preparation, phosphorylation enrichment, quantification, and data analysis strategies permit both targeted and ultra-deep phosphoproteome profiling, but challenges remain in pinpointing biologically relevant phosphorylation events. We describe here technological advances that have facilitated phosphoproteomic analysis of cells, tissues, and biofluids and note applications to neuropathologies in which the phosphorylation machinery may be dysregulated, much as it is in cancer.

EZH2 Modifies Sunitinib Resistance in Renal Cell Carcinoma by Kinome Reprogramming.

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle-regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651-66. ©2017 AACR.

Identification of Plant Kinase Substrates Based on Kinase Assay-Linked Phosphoproteomics.

Protein phosphorylation is one of the key events in the regulation of plant physiological responses to diverse environmental stimuli. As crucial regulators of phosphorylation, protein kinases have been linked to the control of seed germination, flowering, and stress responses. Identifying downstream substrates of kinases is important for dissecting kinase-substrate networks as well as delineating the underlying defense mechanisms in response to extracellular stimulation. Despite the fact that thousands of kinase-substrate networks have been identified in mammalian cells, the downstream substrates of important plant kinases are still elusive. Moreover, it remains challenging to identify bona fide kinase substrates from proteome-wide analyses. Thus, developing methodologies with high sensitivity and specificity is imperative for understanding plant kinase-substrate cascades. Here, we describe a proteomic strategy termed kinase assay-linked phosphoproteomics (KALIP) approach for large-scale identification of the direct substrates of plant kinases with high sensitivity and a low false-positive rate.

Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry.

Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases-casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6-along with 16O4- and 18O4-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%-40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency. Graphical Abstract ᅟ.

Ascl2 inhibits myogenesis by antagonizing the transcriptional activity of myogenic regulatory factors.

Myogenic regulatory factors (MRFs), including Myf5, MyoD (Myod1) and Myog, are muscle-specific transcription factors that orchestrate myogenesis. Although MRFs are essential for myogenic commitment and differentiation, timely repression of their activity is necessary for the self-renewal and maintenance of muscle stem cells (satellite cells). Here, we define Ascl2 as a novel inhibitor of MRFs. During mouse development, Ascl2 is transiently detected in a subpopulation of Pax7+ MyoD+ progenitors (myoblasts) that become Pax7+ MyoD- satellite cells prior to birth, but is not detectable in postnatal satellite cells. Ascl2 knockout in embryonic myoblasts decreases both the number of Pax7+ cells and the proportion of Pax7+ MyoD- cells. Conversely, overexpression of Ascl2 inhibits the proliferation and differentiation of cultured myoblasts and impairs the regeneration of injured muscles. Ascl2 competes with MRFs for binding to E-boxes in the promoters of muscle genes, without activating gene transcription. Ascl2 also forms heterodimers with classical E-proteins to sequester their transcriptional activity on MRF genes. Accordingly, MyoD or Myog expression rescues myogenic differentiation despite Ascl2 overexpression. Ascl2 expression is regulated by Notch signaling, a key governor of satellite cell self-renewal. These data demonstrate that Ascl2 inhibits myogenic differentiation by targeting MRFs and facilitates the generation of postnatal satellite cells.

Identification of Direct Kinase Substrates via Kinase Assay-Linked Phosphoproteomics.

Protein phosphorylation plays an essential role in the regulation of various cellular functions. Dysregulation of phosphorylation is implicated in the pathogenesis of certain cancers, diabetes, cardiovascular diseases, and central nervous system disorders. As a result, protein kinases have become potential drug targets for treating a wide variety of diseases. Identification of kinase substrates is vital not only for dissecting signaling pathways, but also for understanding disease pathologies and identifying novel therapeutic targets. However, identification of bona fide kinase substrates has remained challenging, necessitating the development of new methods and techniques. The kinase assay linked phosphoproteomics (KALIP) approach integrates in vitro kinase assays with global phosphoproteomics experiments to identify the direct substrates of protein kinases. This strategy has demonstrated outstanding sensitivity and a low false-positive rate for kinase substrate screening.

The sensor histidine kinase RgfC affects group B streptococcal virulence factor expression independent of its response regulator RgfA.

Group B streptococci (GBS; Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria that are common asymptomatic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and in certain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number of two-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC and the response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certain GBS clinical isolates, a point mutation in rgfA results in premature truncation of the response regulator. The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that deletion of rgfC in GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with the rgfC mutant increased induction of proinflammatory signaling pathways in vivo. Phosphoproteomic analysis revealed that 19 phosphopeptides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine residues in the rgfC mutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator. Consistent with this observation, microarray analysis of the rgfC mutant indicated that >200 genes showed altered expression compared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associated with GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expression of virulence factors and GBS pathogenesis.