Microbiology and Immunology Graduate Theses (July 2007 - Sept 2016)

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PLEASE NOTE: The discipline of biochemistry is now part of the new interdisciplinary program of Biomedical and Molecular Sciences.

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Regulation of the mexAB-oprM efflux operon of Pseudomonas aeruginosa
Fruci, Michael; Microbiology and Immunology; Poole, R. Keith
Multidrug efflux systems are important determinants of antimicrobial resistance in the human pathogen Pseudomonas aeruginosa where they compromise anti-pseudomonal chemotherapy. The major multidrug efflux system in P. aeruginosa is encoded by the mexAB-oprM operon, expression of which is regulated by a MexR repressor whose activity is negatively modulated by the anti-repressor protein, ArmR. armR occurs as part of a two-gene operon, PA3720-armR, that is regulated by the product of the divergently-transcribed nalC repressor gene, with nalC mutants showing elevated PA3720-armR expression and, so, elevated mexAB-oprM expression and multidrug resistance. The function of the PA3720 protein is unknown. We show here that PA3720 functions as a non-specific RNA binding protein that binds to and destabilizes the adjacent armR-bearing mRNA. Expression of armR is also shown to be translationally coupled to PA3720. In an earlier study, aminoglycosides were shown to induce expression of PA3720-armR, suggesting that these drugs may also promote mexAB-oprM expression dependent on ArmR. Consistent with this, aminoglycosides promoted expression of mexAB-oprM; however, this was independent of ArmR. Instead, the aminoglycoside-responsive AmgRS two-component system mediated aminoglycoside induction of this efflux system. Consistent with this, mutational activation of the AmgS sensor kinase yielded elevated levels of mexAB-oprM expression, and purified AmgR bound specifically to the mexAB-oprM promoter region. Screening of P. aeruginosa strain PAO1 transposon mutants for increased sensitivity to carbenicillin- carbenicillin is a MexAB-OprM substrate and carbenicillin sensitivity is a surrogate for loss of mexAB-oprM expression - led to the identification of several genes with probable roles in mexAB-oprM expression or activity. The gene disrupted in one of these mutants, bamB, encodes for a component of the outer membrane β-barrel protein assembly machinery and was assessed for its role in facilitating the assembly of OprM, a β-barrel outer membrane protein. Western immunoblotting revealed that BamB is not required for OprM insertion into the outer membrane. These studies provide additional insights into the regulation of mexAB-oprM expression in P. aeruginosa.
Development of models to study Salmonella enterica infection of THP-1 cells pre-treated with IL-27
(2015-11-03) Plater, Samantha; Microbiology and Immunology; Martin, Nancy L.
IL-27 has been shown to have important pro- and anti-inflammatory roles in the adaptive immune response. However, its role in the innate immune response is less well characterized. The goal of this research was to examine the role of IL-27 in the innate immune response. Two in vitro models of Salmonella enterica serovar Typhimurium infection of the human monocytic cell line, THP-1, were developed. S. typhimurium is an intracellular pathogen that can facilitate its own uptake into host cells where it is able to replicate, often leading to host cell death. Based upon previous research on the role of IL-27 in innate immunity, it was hypothesized that IL-27 pre-treatment of THP-1 cells would result in an enhanced ability of THP-1 cells to fight S. typhimurium infection. Infecting THP-1 cells with late stationary phase S. typhimurium resulted in a low level of infection whereas infection of THP-1 cells with late exponential phase S. typhimurium led to a high level of THP-1 cell infection. Adherent THP-1 cells were stimulated with IL-27 for 16 hours prior to infection with either late stationary or late exponential phase S. typhimurium. A gentamicin protection assay was used to quantify internalized bacteria to assess if IL-27 pre-treatment affected the initial invasion and subsequent survival of S. typhimurium in THP-1 cells. THP-1 cells stimulated with IL-27 had the same level of internalized S. typhimurium as untreated THP-1 cells initially and over the subsequent 12 hours. THP-1 cell viability was monitored between 1-12 hours after the addition of S. typhimurium to determine if IL-27 pre-treatment influenced THP-1 cell death. Pre-treatment with IL-27 did not result in a difference in THP-1 cell death. TNFα and IL-6 secretion from S. typhimurium infected THP1 cells were assayed to determine if IL-27 pre-treatment resulted in an altered cytokine response. IL-27 stimulation resulted in significantly higher (p<0.01) levels of TNFα and IL-6 than untreated cells at 1-12 hours after the addition of S. typhimurium indicating IL-27 stimulation of THP-1 cells promotes a pro-inflammatory response. Overall, IL-27 pre-treatment of THP-1 cells did not alter S. typhimurium infection of THP-1 cells but did enhance the pro-inflammatory response by these cells indicating IL-27 has a role in the pro-inflammatory response by innate immune system to S. typhimurium infection. Further work is needed to explore the host-pathogen interaction that resulted in enhanced cytokine production in THP-1 cells and if this plays a significant role in immune cell survival over an extended time frame. Understanding the role of IL-27 in the innate immune response to pathogens will provide more information on how the immune system combats disease.
Regulation of the mexCD-oprJ multidrug efflux operon in Pseudomonas aeruginosa: characterization of the NfxB-EsrC repressor interaction
(2015-09-26) Mikalauskas, Alaya; Microbiology and Immunology; Poole, R. Keith
The MexCD-OprJ multidrug efflux pump of Pseudomonas aeruginosa is a RND-type efflux system that is capable of exporting, and thus providing resistance to, multiple classes of clinically-relevant antibiotics such as fluoroquinolones, β-lactams and macrolides. Although quiescent in wild-type P. aeruginosa, mexCD-oprJ is induced by membrane-damaging agents such as detergents and the biocide chlorhexidine (CHX), which perturb the cell envelope. Induction of mexCD-oprJ by membrane-damaging agents requires the envelope stress sigma factor AlgU, an indication that MexCD-OprJ is a component of the envelope stress response in P. aeruginosa. In the absence of CHX-mediated envelope stress, the expression of mexCD-oprJ is regulated solely by the NfxB repressor, which binds as a tetramer to the nfxB-mexC intergenic region to repress transcription of the mexCD-oprJ operon. Recently, a second repressor of mexCD-oprJ with homology to NfxB, EsrC, was discovered. In the absence of CHX-mediated envelope stress, the expression of esrC is negatively regulated by NfxB. In the presence of CHX-mediated envelope stress, the repressor activity of NfxB is alleviated and EsrC is produced, where it contributes to moderate repression of the mexCD-oprJ operon, which is expressed at moderate levels. Interestingly, EsrC requires NfxB in order to act as a repressor of the mexCD-oprJ operon. Consistent with previous research, this study demonstrated a physical interaction between NfxB and EsrC. In addition, this study showed that the C-terminal domains of NfxB and EsrC are required for the NfxB-EsrC interaction to occur. The demonstration of an interaction between NfxB and EsrC suggests that these two repressors co-regulate the expression of the mexCD-oprJ operon in the presence of envelope stress to either prevent the potentially detrimental effects of MexCD-OprJ overexpression, or to modulate mexCD-oprJ expression in response to multiple envelope stress signals which are recognized by the NfxB and EsrC components of the heteromultimeric repressor.
Characterizing the Role of the Herpes Simplex Virus Type 2 UL21 Protein in Viral Nuclear Egress
(2015-07-17) Nassiri, Arash; Microbiology and Immunology; Banfield, Bruce
The herpes simplex virus type 2 (HSV-2) is an important human pathogen that is the main cause of genital herpes infections and has a significant and wide-ranging impact on human health. HSV-2 infections are one of the most common sexually transmitted diseases and are highly prevalent worldwide, thus making understanding their infection mechanisms all the more relevant. The UL21 gene, which is conserved amongst members of the Alphaherpesvirinae subfamily, encodes a tegument protein that is essential for HSV-2 propagation. More specifically, UL21 plays a critical role in the primary envelopment of capsids during nuclear egress, however the precise mechanism by which it does this is unclear. To investigate the role of UL21 in HSV-2 nuclear egress, we focused on events upstream of primary envelopment. First, we examined the role of UL21 in disrupting the localization of nuclear lamins during infection. Nuclear rim localization of lamins A, C, B1, and B2 was indistinguishable between the wild-type (WT), UL21 null (∆UL21), or repaired (∆UL21R) HSV-2 strains, suggesting UL21 is not implicated in the disassembly of nuclear lamins at the nuclear membrane. Additionally, UL21 did not influence the localization of the nuclear egress complex (NEC), composed of UL31 and UL34, insofar as cellular and virally-encoded UL31 and UL34 localization patterns were indistinguishable between the UL21 null, and repaired or wild-type strains. Lastly, in an attempt to characterize interacting partners for UL21, we focused on proteins implicated in nuclear egress or proteins that may function to recruit UL21 to the nuclear membrane, where it may carry out its essential role(s). While no interactions were detected between UL21 and key mediators of primary envelopment including UL31, UL34, or Us3, UL21 localization at the nuclear rim was enhanced by the overexpression of the inner nuclear membrane protein, LAP2β, suggesting a potential interaction between the two proteins. However, more experiments are warranted before any conclusive remarks can be made regarding such interaction. Taken together, these data have shed light on HSV-2 nuclear egress and have eliminated possible theories regarding the role of UL21 in HSV-2 primary envelopment.
Identification of Novel Genes Involved in N-Glycosylation and Regulation of Archaella Assembly and Exploration of the Effect of N-Glycosylation on Archaella Assembly in Methanococcus Maripaludis
(2015-07-17) Ding, Yan; Microbiology and Immunology; Jarrell, Ken F.
In this thesis, using Methanococcus maripaludis as a model organism, significant, novel advances have been made to the understanding of the archaeal N-glycosylation pathway as well as archaella regulation and assembly. The archaellum is the swimming organelle in the third domain of life, the Archaea. Genes involved in archaellation are mainly clustered in the fla operon. The structural proteins of archaella, termed archaellins, are usually glycoproteins modified with N-glycans. In M. maripaludis, a euryarchaeon, all three archaellins FlaB1, FlaB2 and FlaB3 are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is a unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-L-erythro-hexos-5-ulo-1,5-pyranose exclusively found in this species. Using FlaB2 as a reporter protein, I genetically identified three novel genes, aglU, aglV and aglW, which are involved in the synthesis of the archaellin N-glycan. AglU, a putative threonine transferase, is thought to be responsible for the transfer of the threonine onto the third sugar residue in the N-glycan. AlgV and AglW, annotated as a methyltransferase and a UDP-glucose 4-epimerase, respectively, are involved in the biosynthesis of the unique terminal sugar. Since it has been shown that disruption of the N-glycosylation pathway caused defects in archaella assembly or function, I explored the specific requirement of N-glycosylation of archaellins on archaellation by eliminating the four N-glycosylation sites in FlaB2 in all possible combinations. As many as three N-glycosylation sites could be removed from FlaB2 without interfering with archaella assembly or function. However, archaella could not be assembled when all four N-glycosylation sites in FlaB2 are eliminated. While there is evidence that archaella synthesis in M. maripaludis is not constitutive, no transcriptional regulators had been identified for the fla operon in any euryarchaeon. I genetically identified and characterized ArnE as the first euryarchaeotic transcriptional activator of the fla operon. Deletion of arnE resulted in the cessation of the transcription and translation of the reporter gene flaB2 and translation of FlaB2 was restored when the mutant was complemented with arnE in trans. Electrophoretic mobility shift assays confirmed the specific binding of purified ArnE to DNA fragments from the fla promoter.

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