Cell Biology & Molecular Genetics
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Item INVESTIGATION OF CYCLIC DINUCLEOTIDE HOMEOSTASIS AND THE HYDROLYSIS OF THEIR LINEAR INTERMEDIATES IN BACTERIA(2019) Weiss, Cordelia Anne; Winkler, Wade C; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The synthesis of cyclic dinucleotides as signals is one strategy bacteria use to sense and adjust to environmental changes. Cyclases synthesize the cyclic dinucleotide, while phosphodiesterases cleave it to yield a linear diribonucleotide, which is recycled into monoribonucleotides by other enzymes. For many bacteria, cyclic di-GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile community. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. Bacillus subtilis is a model for the study of bacterial differentiation, yet how c-di-GMP functions in this organism is not fully understood. This work began with construction of a fluorescent reporter to measure c-di-GMP abundance in B. subtilis, which showed that c-di-GMP levels are strikingly different among differentiated subpopulations. These data highlight how single-cell approaches can be used to analyze metabolic trends within bacterial populations and demonstrate that for some bacteria, c-di-GMP levels are adjusted heterogeneously across bulk populations. The enzymes Orn, NrnA, NrnB, and NrnC have been proposed to act as general 3’-5’ exoribonucleases that preferentially process ‘short’ oligoribonucleotides. Intriguingly, Orn also performs a crucial role in c-di-GMP homeostasis by processing the pGpG generated from c-di-GMP production. To discover the molecular basis for Orn’s ability to ‘select’ short RNAs, and to elucidate the relationship between Orn and the diribonucleotide pGpG, we combined structural, biochemical, and in vivo analyses of RNA cleavage. These data reveal that Orn is not a general exoribonuclease of short RNA oligoribonucleotides, as previously believed, but instead acts as a dedicated ‘diribonucleotidase’. Our studies indicate RNA degradation as a step-wise process with a dedicated enzyme for the clearance of diribonucleotides, which affect cellular physiology and viability. Examination of the roles of NrnA and NrnB is underway. We conducted an initial study to determine if NrnA and NrnB are redundant proteins, as has been proposed, and if they might also act as ‘diribonucleotidases’. These data show that they exhibit different substrate preferences and that they may have unique cellular functions. Therefore this work changes the perception of the role(s) Orn plays and that a re-evaluation of ‘short’ RNases is needed.Item NEISSERIA GONORRHOEAE MODULATES INFECTIVITY BASED ON PROPERTIES OF HUMAN CERVICAL EPITHELIA AND PHASE VARIABLE BACTERIAL SURFACE STRUCTURES(2019) Yu, Qian; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) infection in the human female reproductive tract causes various clinical outcomes, from no symptom to severe complications. The major barrier to a better understanding of GC infection in women is the lack of experimental system closely mimicking in vivo infection. Here, I developed a human cervical tissue explant model, which maintains the heterogeneity of the cervical epithelium. Using this model, my thesis research examined the impact of the heterogeneity of the cervical epithelium and the phase variation of GC surface structures on GC infectivity. My research revealed that GC preferentially colonize the ectocervix and the transformation zone (TZ), but exclusively penetrate into the subepithelial tissues of the TZ and endocervix. Pili are essential for GC colonization in all regions of the cervix. Expression of Opa isoforms that bind to the host receptors CEACAM (OpaCEA) enhances GC colonization in the ecto/endocervix but inhibits GC penetration into the endocervix. However, GC infectivity in the TZ does not respond to Opa phase variation, due to the low expression level and intracellular location of CEACAMs in the TZ epithelial cells. OpaCEA enhances GC colonization in the ecto/endocervix by inhibiting epithelial exfoliation and suppresses GC penetration into the endocervical subepithelium by inhibiting GC-induced disassembly of the apical junction. Opa-mediated modulation of GC infectivity depends on the immune receptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 and its downstream phosphatase SHP. The effect of epithelial cell polarity on GC invasion was studied using a cell line model. My results show that GC invade more efficiently into non-polarized than polarized epithelial cells without changing the adhesion efficiency. Opa (phase variable) expression enhances both adhesion and invasion in both non-polarized and polarized cells. In non-polarized cells, Opa expression induces F-actin accumulation and microvilli elongation underneath GC microcolonies, suggesting an actin-mediated uptake of GC. In contrast, GC expressing no Opa reduce F-actin and demolish microvilli underneath microcolonies in both polarized epithelial cell line and endocervical epithelial cells potentially by increasing calcium flux, NMII activation and the redistribution of actin nucleation factor Arp2/3 from the apical surface. Taking together, my research demonstrates that both the heterogeneity of the cervical epithelium and the phase variation of bacterial surface structures regulate GC infectivity in the human cervix, either dominated by colonization or penetration, consequently influencing the clinical outcomes of the infection.Item Structure-Guided Engineering of a Multimeric Bacteriophage-Encoded Endolysin PlyC(2019) Shang, Xiaoran; Nelson, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Emerging antibiotic resistance has become a global health threat. One alternative to antibiotics is bacteriophage-encoded endolysins. Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage replication cycle resulting in bacterial cell lysis and progeny bacteriophage release. Endolysins are also capable of destroying the Gram-positive bacterial peptidoglycan when applied externally as recombinant proteins. These enzymes typically consist of an enzymatically active domain (EAD) and a separate cell wall binding domain (CBD). Studies have shown therapeutic efficacy of endolysins in vitro and in vivo, with no resistance developed to date. An endolysin from the streptococcal C1 phage, known as PlyC, has the highest activity of any endolysin reported. It also has a unique multimeric structure consisting of one activity subunit (PlyCA) harboring two synergistically acting catalytic domains, GyH and CHAP, and eight identical binding subunits (PlyCB) forming an octameric ring. Groups A, C, and E streptococci as well as Streptococcus uberis are sensitive to the lytic activities of PlyC. In order to harness the potent activity of PlyC for use against other bacteria, we sought to change/extend the host range of PlyC by engineering PlyCB and PlyCA, respectively. We first used a structure-guided mutagenesis method to obtain the single PlyCB monomer subunit, PlyCBK40A E43A (PlyCBm), aiming to study the binding mechanism of PlyCB. Via fluorescence microscopy and binding assays, we determined that PlyCBm retained the host range of the octamer with a much lower binding affinity, which suggests the PlyCB octamer binds concurrently to a specific epitope on the bacterial surface resulting in a tight, stable interaction. Thus, it is not feasible to change/extend the PlyC host range via engineering PlyCB. Next, we proposed a novel design to engineer PlyCA. We successfully created two chimeric endolysins, ClyX-1 and ClyX-2, possessing the synergistic activity of the GyH and CHAP catalytic domains, but extended the host range to include, Streptococcus pneumoniae, Group B streptococci, Streptococcus mutans, and Enterococcus faecalis, all strains previously insensitive to PlyC. Finally, we tested a novel hypothesis that a positively charged catalytic domain could display lytic activity in a CBD-independent manner resulting in a broad host range. Using the PlyC CHAP domain as a model, we converted the net surface charge of the CHAP domain from negative three to positive one through positive seven. Notwithstanding the range of charges, our mutant CHAP domains did not show lytic activity in a CBD-independent manner, suggesting that other factors, like surface charge distribution, need to be considered in such a way of engineering.Item Discovery and Characterization of Antiterminator Proteins in Bacteria(2018) Goodson, Jonathan Ryan; Winkler, Wade; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Transcription is a discontinuous process, where each nucleotide incorporation cycle offers a decision between elongation, pausing, halting, or termination. In bacteria, many regulators—including protein antiterminators or cis-acting regulatory RNAs, such as riboswitches—exert their influence over transcription elongation. Through such mechanisms, these regulators can couple physiological or environmental signals to transcription attenuation, a process where RNA structure directly influences formation of transcription termination signals. However, through another regulatory mechanism called processive antitermination (PA), RNA polymerase can become induced to bypass termination sites over much greater distances than transcription attenuation can offer. These mechanisms are widespread in bacteria, although only a few mechanistic classes have been discovered overall. The aim of the research in this dissertation is two-fold: to identify novel genetic regulatory mechanisms targeting transcription termination and to systematically study the diversity and breadth distribution of these mechanisms among bacteria. This research focuses on two distinct mechanisms, each representing one of these mechanisms of antitermination. First, I detail discovery of LoaP, a specialized paralog of the universally conserved NusG transcription elongation factor. Our data demonstrate that Bacillus velezensis LoaP controls gene expression of antibiotic biosynthesis gene clusters by promoting readthrough of transcription termination sites. Additionally, we show that, unlike other bacterial NusG proteins, LoaP binds RNA with high affinity, and with apparent specificity for a sequence in the 5′ leader regions of its target operons. Second, we describe the interaction between a family of antitermination proteins containing the ANTAR RNA-binding domain with its target RNA. We show that ANTAR-containing proteins bind a tandem stem-loop RNA motif to prevent formation of terminator structures. Using a combination of mutagenesis strategies, we elucidate some of the RNA-binding requirements of a representative ANTAR protein. Finally, employed bioinformatic and phylogenetic approaches to place these regulators in the context of their entire protein families, learning about the distribution of these mechanisms, their association with particular potential regulons, and sequence composition of different protein subfamilies.Item STIMULATION OF GROWTH AND METABOLITES PRODUCTION OF LACTOBACILLUS IN CONTROL OF ENTERIC BACTERIAL PATHOGEN INFECTION AND IMPROVING GUT HEALTH(2018) Peng, Mengfei; Biswas, Debabrata; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Foodborne enteric diseases cause millions of illness and thousands of deaths annually in the United States. Major enteric bacterial pathogens include Salmonella, enterohemorrhagic Escherichia coli O157:H7 (EHEC), Campylobacter, Listeria, Shigella, Vibrio, and Yersinia which account for more than 90% cases of culture-confirmed infections. Among these causative agents, Salmonella enterica is responsible for the highest rate of hospitalization and EHEC has the lowest infectious dose. Their pathogenesis involves numerous virulent factors whereas their colonization and invasion on host gut intestine mainly depend on the type III secretion system. The prevention of foodborne enteric diseases is of great concern to public health professionals, farmers, and food producers. Due to the increased public health concern about antibiotic-resistance dissemination, alternative strategies such as pro-commensal approach by applying probiotics, prebiotics, and combination of both (synbiotics) are of interests for prevention and therapy of foodborne enteric diseases. In this study, we both in vitro and in vivo evaluated the preventive capabilities of Lactobacillus against enteric pathogenic bacterial colonization and infection. Functional food cocoa and peanut containing prebiotic-like ingredients selectively promoted the growth of beneficial bacteria and stimulated the production of bio-active metabolites especially conjugated linoleic acids in Lactobacillus. We also detected the synergistic effects of Lactobacillus and cocoa/peanut on competitive exclusion of S. Typhimurium and EHEC, alteration on physicochemical properties, disruption of host-pathogen interactions, and down-regulation on virulence gene expressions. Furthermore, with homologous recombination, we overexpressed myosin cross-reactive antigen gene encoding linoleate isomerase in L. casei and improved the efficiency in their linoleic acids production as well as the gut intestinal adherence and colonization. By applying genetically engineered LC-CLA, S. Typhimurium and EHEC were much effectively controlled and restricted from all aspects in vitro mentioned before. Additionally, the in vivo pre-administration of LC-CLA reduced S. Typhimurium gut intestinal colonization/infection in a significant level and induced anti-inflammatory effects, which benefitted the overall mice gut health. Our findings established a baseline upon which self-promoting probiotic independent from prebiotic in prevention or treatment against enteric diseases can be explored.Item CHARACTERIZING THE INHIBITION OF INNATE IMMUNE SIGNALING BY MYCOBACTERIUM TUBERCULOSIS(2017) Ahlbrand, Sarah; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Numerous cytokines are induced as a consequence of infection by Mycobacterium tuberculosis (Mtb), an intracellular pathogen that is responsible for millions of deaths each year. These cytokines play varied roles in eliciting and regulating the innate and adaptive immune responses against Mtb and often determine disease outcome. The pro-inflammatory cytokine interleukin 1β (IL-1β) is required for Mtb maintenance and clearance, as mice lacking the ability to produce IL-1β succumb very quickly to infection compared to infected wildtype mice. In contrast, interferon β (IFNβ) is largely thought to be detrimental to the host during Mtb infection based on collective studies in patients and mouse models. Both IL-1β and IFNβ are induced during Mtb infection, but here we demonstrate that Mtb has also evolved mechanisms to reduce their production and/or signaling. Previously, we’ve shown that Mtb can inhibit IL-1β production induced by the Absent in Melanoma 2 (AIM2) inflammasome. These findings were expanded upon by investigating the mechanisms by which Mtb inhibits AIM2 activation. A gain-of-function screen was also utilized in the non-pathogenic mycobacterial species Msmeg to identify Mtb genomic regions contributing to this phenomenon. In addition, we demonstrate that Mtb inhibits IFNβ signaling by inhibiting IFNβ-induced JAK1 and Tyk2 phosphorylation, leading to changes in the host type I interferon transcriptional profile. These studies provide insight into two previously undescribed mechanisms Mtb utilizes to manipulate the host immune response.Item CHARACTERIZING THE ROLE OF THE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM ENZYME II LOCI IN THE PATHOGENESIS OF THE GROUP A STREPTOCOCCUS(2017) Sundar, Ganesh; McIver, Kevin S; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)-mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared with parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC-encoding genes in the GAS MGAS5005 genome to functionally characterize each EIIC. It was found that a few EIIs had a limited in uence on PTS sugar metabolism, whereas others were promiscuous. The mannose-speci c EII locus exhibited the most in uence on PTS sugar metabolism. Importantly, the mannose-speci c EII also acted to prevent the early onset of SLS-mediated hemolysis. These roles were not identical in two different M1T1 GAS strains, highlighting the versatility of the PTS to adapt to strain-speci c needs. This is further illustrated by the fructose-speci c EII, which is important for survival in whole human blood for MGAS5005, but not 5448. The mannose-speci c EII can transport glucose in other pathogens, but the route of glucose utilization is unknown in GAS. MGAS5005 mutants were generated in a non-PTS glucose transporter (GlcU) and a glucokinase (NagC) of an annotated non-PTS glucose metabolic pathway. Since ∆ptsI, ∆nagC, and ∆glcU all grow to some extent in glucose, it is evident that glucose can be metabolized both by PTS and non-PTS routes. . However, the route of glucose utilization affects overall pathogenesis, as ∆nagC survives like WT in whole human blood, whereas ptsI is unable to survive. Subcutaneous infection of mice with ∆nagC did not exhibit increased lesion size, although these lesions are more severe than MGAS5005 due to the early onset of hemolysis. Overall this suggests that the routes of glucose metabolism greatly in uence SLS-mediated hemolysis. These results highlight that PTS carbohydrate metabolism plays an important role for GAS pathogenesis in both the skin and whole human blood, through the actions of EIIs.Item AN INVESTIGATION ON A BACTERIOPHAGE ENDOLYSIN POSSESSING ANTIMICROBIAL ACTIVITY AGAINST ANTIBIOTIC-RESISTANT STAPHYLOCOCCUS AUREUS(2016) Linden, Sara Beth; Nelson, Daniel C; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Staphylococcus aureus is one of the most common causes of nosocomial (i.e. hospital-acquired) infection. Significantly, over 90% of S. aureus strains are resistant to penicillin, and since the mid-1980’s, methicillin-resistant S. aureus (MRSA) strains have become prevalent in hospitals worldwide, with resistance rates approaching 70%. In the U.S. alone, MRSA is responsible for over 100,000 invasive life threatening infections, such as necrotizing fasciitis, and causes 20,000 deaths annually. More worrisome, a variant known as community-acquired MRSA (CA-MRSA) is spreading in schools, gymnasiums, and even professional sports teams, where it infects otherwise healthy adolescents and young adults. Vancomycin is often considered the last antibiotic of choice against MRSA and other Gram-positive pathogens. However, rates of vancomycin-resistant enterococci (VRE) have already reached 30% and it is widely believed that emergence of vancomycin-resistant S. aureus (VRSA) is due to gene transfer during co-colonization of MRSA and VRE. Thus, alternative antimicrobial approaches are desperately needed. Endolysins, or peptidoglycan hydrolases, are phage-derived enzymes that actively lyse bacterial cells upon direct contact and may be considered such an alternative option. Moreover, the inability of bacteria to evolve resistance to endolysins is due to the specificity of the N-terminal catalytic domain, which cleaves a conserved peptidoglycan bond, and the C-terminal cell wall binding domain, which binds a cell surface moiety. This thesis represents an investigation into the endolysin PlyGRCS, which displays potent bacteriolytic activity against all antibiotic-resistant strains of S. aureus tested. This enzyme is active in physiologically relevant conditions (pH, NaCl, temperature), and its activity is greatly enhanced in the presence of calcium. PlyGRCS is the first endolysin with a single catalytic domain that cleaves two distinct sites in the peptidoglycan. Unlike antibiotics, PlyGRCS displays anti-biofilm activity, preventing, removing, and killing biofilms grown on abiotic and biotic surfaces. Engineering efforts were made to create an enzyme with a variable binding domain, which unfortunately displayed less activity than the wild type endolysin in the conditions tested. The antimicrobial efficacy of PlyGRCS was validated in a mouse model of S. aureus septicemia. The results from this study indicate that the endolysin PlyGRCS is a revolutionary therapeutic that should be further pursued for subsequent translational development.Item A SUBSET OF 3´ TO 5´ EXORIBONUCLEASES ARE THE PRIMARY ENZYMES RESPONSIBLE FOR THE DEGRADATION OF PGPG IN CYCLIC DI-GMP SIGNALING(2016) Orr, Mona; Lee, Vincent T; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bis-(3´-5´)-cyclic dimeric guanosine monophosphate, or cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that regulates processes such biofilm formation, motility, and virulence. C-di-GMP is synthesized by diguanylate cyclases (DGCs), while phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMPs by previously unidentified enzymes termed PDE-Bs. To identify the PDE-B responsible for pGpG turnover, a screen for pGpG binding proteins in a Vibrio cholerae open reading frame library was conducted to identify potential pGpG binding proteins. This screen led to identification of oligoribonuclease (Orn). Purified Orn binds to pGpG and can cleave pGpG to GMP in vitro. A deletion mutant of orn in Pseudomonas aeruginosa was highly defective in pGpG turnover and accumulated pGpG. Deletion of orn also resulted in accumulation c-di-GMP, likely through pGpG-mediated inhibition of the PDE-As, causing an increase in c-di-GMP-governed auto-aggregation and biofilm. Thus, we found that Orn serves as the primary PDE-B enzyme in P. aeruginosa that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway. However, not all bacteria that utilize c-di-GMP signaling also have an ortholog of orn, suggesting that other PDE-Bs must be present. Therefore, we asked whether RNases that cleave small oligoribonucleotides in other species could also act as PDE-Bs. NrnA, NrnB, and NrnC can rapidly degrade pGpG to GMP. Furthermore, they can reduce the elevated aggregation and biofilm formation in P. aeruginosa ∆orn. Together, these results indicate that rather than having a single dedicated PDE-B, different bacteria utilize distinct RNases to cleave pGpG and complete c-di-GMP signaling. The ∆orn strain also has a growth defect, indicating changes in other regulatory processes that could be due to pGpG accumulation, c-di-GMP accumulation, or another effect due to loss of Orn. We sought to investigate the genetic pathways responsible for these growth defect phenotypes by use of a transposon suppressor screen, and also investigated transcriptional changes using RNA-Seq. This work identifies that c-di-GMP degradation intersects with RNA degradation at the point of the Orn and the functionally related RNases.Item FUNCTIONAL CHARACTERIZATION OF THE LINK BETWEEN CARBOHYDRATE METABOLISM AND THE PATHOGENESIS OF THE INVASIVE M1T1 GROUP A STREPTOCOCCUS(2016) Valdes, Kayla Maureen; McIver, Kevin S; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Group A Streptococcus (GAS), or Streptococcus pyogenes, is a strict human pathogen that colonizes a variety of sites within the host. Infections can vary from minor and easily treatable, to life-threatening, invasive forms of disease. In order to adapt to niches, GAS utilizes environmental cues, such as carbohydrates, to coordinate the expression of virulence factors. Research efforts to date have focused on identifying how either components of the phosphoenolpyruvate-phosphotransferase system (PTS) or global transcriptional networks affect the regulation of virulence factors, but not the synergistic relationship between the two. The present study investigates the role of a putative PTS-fructose operon encoded by fruRBA and its role in virulence in the M1T1 strain 5448. Growth in fructose resulted in induction of fruRBA. RT-PCR showed that fruRBA formed an operon, which was repressed by FruR in the absence of fructose. Growth and carbon utilization profiles revealed that although the entire fruRBA operon was required for growth in fructose, FruA was the main fructose transporter. The ability of both ΔfruR and ΔfruB mutants to survive in whole human blood or neutrophils was impaired. However, the phenotypes were not reproduced in murine whole blood or in a mouse intraperitoneal infection, indicating a human-specific mechanism. While it is known that the PTS can affect activity of the Mga virulence regulator, further characterization of the mechanism by which sugars and its protein domains affect activity have not been studied. Transcriptional studies revealed that the core Mga regulon is activated more in a glucose-rich than a glucose-poor environment. This activation correlates with the differential phosphorylation of Mga at its PTS regulatory domains (PRDs). Using a 5448 mga mutant, transcriptome studies in THY or C media established that the Mga regulon reflects the media used. Interestingly, Mga regulates phage-encoded DNases in a low glucose environment. We also show that Mga activity is dependent on C-terminal amino acid interactions that aid in the formation of homodimers. Overall, the studies presented sought to define how external environmental cues, specifically carbohydrates, control complex regulatory networks used by GAS, contribute to pathogenesis, and aid in adaptation to various nutrient conditions encountered.