PhoA gene fusions in Legionella pneumophila generated in vivo using a new transposon, MudphoA

To enable effective use of phoA gene fusions in Legionella pneumophila , we constructed MudphoA , a derivative of the mini-Mu phage Mu dII4041, which is capable of generating gene fusions to the Escherichia coli alkaline phosphatase gene (EC 3.1.3.1). Although an existing fusion-generating transposon, TnphoA , has been a useful tool for studying secreted proteins in other bacteria, this transposon and other Tn 5 derivatives transpose inefficiently in Legionella pneumophila , necessitating the construction of a more effective vector for use in this pathogen. Using MudphoA we generated fusions to an E. coli gene encoding a periplasmic protein and to an L. pneumophila gene encoding an outer membrane protein; both sets of fusions resulted in alkaline phosphatase activity. We have begun to use MudphoA to mutate secreted proteins of L. pneumophila specifically, since this subset of bacterial proteins is most likely to be involved in host-bacterial interactions. This modified transposon may be useful for studies of other bacteria that support transposition of Mu, but not Tn 5 , derivatives.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75760/1/j.1365-2958.1992.tb01355.x.pd

Progress in the pathogenesis of Legionella pneumophila

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29516/1/0000603.pd

Growth of Legionella pneumophila in a human macrophage-like (U937) cell line

We established a model of the bacteria-macrophage interaction to study the cellular basis of Legionella pneumophila pathogenesis and to characterize avirulent L. pneumophila. We found that U937 cells, which are derived from a human histiocytic lymphoma cell line, support intracellular growth of L. pneumophila with a doubling time of 6 h, and that sustained intracellular growth is associated with a cytopathic effect (CPE) that can be detected by microscopic examination and quantified with the vital stain 3-(4,5-dimethyl thiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (MTT). An L. pneumophila isolate obtained directly from infected guinea-pig spleens can grow and produce CPE in these cells, destroying most of the cell layer after 72 h of growth. Only 106 organisms of this strain are required to kill 50% of guinea-pigs inoculated by the intraperitoneal route. In contrast, an avirulent isolate derived by 203 successive plate passages of the same strain can neither kill guinea-pigs at an intraperitoneal inoculum of 107 nor grow or produce CPE in U937 cells. Since the cells were able to differentiate between a virulent and an avirulent strain of L. pneumophila, we conclude that U937 cells are an appropriate model system for study of the bacteria-macrophage interaction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27184/1/0000187.pd

Transfer and maintenance of small, mobilizable plasmids with ColE1 replication origins in Legionella pneumophila

With the mutagenesis of specific, virulence-associated genes of Legionella pneumophila as the eventual goal, methods for gene transfer to these bacteria were developed. Following the observations of others that conjugative, broad-host-range plasmids could be transferred from Escherichia coli to L. pneumophila at low frequency, we constructed a small mobilizable vector, pTLP1, which carries oriV from pBR322, oriT from pRK2, Kmr from Tn5, and an L. pneumophila-derived fragment to permit chromosomal integration. In triparental matings including an E. coli with a conjugative (Tra+) helper plasmid, kanamycin-resistance was transferred from E. coli to L. pneumophila. Southern hybridization of L. pneumophila transconjugants showed that pTLP1 was replicated autonomously. Additional matings of plasmids having deletions or substitutions of pTLP1 sequences confirmed that replication in L. pneumophila requires oriV only. pTLP1 was maintained in L. pneumophila with passage on medium containing kanamycin but was rapidly lost after passage on nonselective medium. This plasmid instability in L. pneumophila is most likely due to rapid generation of plasmid-free segregants because of plasmid multimerization and low plasmid copy number. We conclude that mobilizable pBR322-derived plasmids can be used as shuttle vectors to transfer cloned genes to L. pneumophila, a feature that can be exploited for the purposes of mutagenesis or genetic complementation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27233/1/0000240.pd

Site-specific mutagenesis in Legionella pneumophila by allelic exchange using counterselectable ColE1 vectors

To study the molecular pathogenesis of infection by Legionella pneumophila, a technique of site-specific mutagenesis by allelic exchange was evaluated. To develop this system, we optimized conjugal DNA transfer by isolating a mutant that functions 1000-fold more efficiently as a recipient than the wild type strain, identified two counter-selectable markers, rpsL and sacB, that function in L. pneumophila, and constructed a counterselectable Co1E1 vector. Allelic exchange of a L. pneumophila chrosomal gene was achieved at a frequency of 10-5 per transconjugant. The allelic exchange procedure itself did not alter the ability of L. pneumophila to infect macrophages, indicating that the system can be used to study this aspect of virulence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27030/1/0000018.pd

CovR-Controlled Global Regulation of Gene Expression in Streptococcus mutans

CovR/S is a two-component signal transduction system (TCS) that controls the expression of various virulence related genes in many streptococci. However, in the dental pathogen Streptococcus mutans, the response regulator CovR appears to be an orphan since the cognate sensor kinase CovS is absent. In this study, we explored the global transcriptional regulation by CovR in S. mutans. Comparison of the transcriptome profiles of the wild-type strain UA159 with its isogenic covR deleted strain IBS10 indicated that at least 128 genes (∼6.5% of the genome) were differentially regulated. Among these genes, 69 were down regulated, while 59 were up regulated in the IBS10 strain. The S. mutans CovR regulon included competence genes, virulence related genes, and genes encoded within two genomic islands (GI). Genes encoded by the GI TnSmu2 were found to be dramatically reduced in IBS10, while genes encoded by the GI TnSmu1 were up regulated in the mutant. The microarray data were further confirmed by real-time RT-PCR analyses. Furthermore, direct regulation of some of the differentially expressed genes was demonstrated by electrophoretic mobility shift assays using purified CovR protein. A proteomic study was also carried out that showed a general perturbation of protein expression in the mutant strain. Our results indicate that CovR truly plays a significant role in the regulation of several virulence related traits in this pathogenic streptococcus

Activation of the SMU.1882 Transcription by CovR in Streptococcus mutans

In Streptococcus mutans, the global response regulator CovR plays an important role in biofilm formation, stress-tolerance response, and caries production. We have previously shown that CovR acts as a transcriptional repressor by binding to the upstream promoter regions of its target genes. Here, we report that in vivo, CovR activates the transcription of SMU.1882, which encodes a small peptide containing a double-glycine motif. We also show that SMU.1882 is transcriptionally linked to comA that encodes a putative ABC transporter protein. Several genes from man gene clusters that encode mannose phosphotranferase system flank SMU.1882 -comA genes. Genomic comparison with other streptococci indicates that SMU.1882 is uniquely present in S. mutans, while the man operon is conserved among all streptococci, suggesting that a genetic rearrangement might have taken place at this locus. With the use of a transcriptional reporter system and semi-quantitative RT-PCR, we demonstrated the transcriptional regulation of SMU.1882 by CovR. In vitro gel shift and DNase I foot-printing analyses with purified CovR suggest that CovR binds to a large region surrounding the -10 region of the P1882. Using this information and comparing with other CovR regulated promoters, we have developed a putative consensus binding sequence for CovR. Although CovR binds to P1882, in vitro experiments using purified S. mutans RpoD, E. coli RNA polymerase, and CovR did not activate transcription from this promoter. Thus, we speculate that in vivo, CovR may interfere with the binding of a repressor or requires a cofactor

Incompetence of Neutrophils to Invasive Group A streptococcus Is Attributed to Induction of Plural Virulence Factors by Dysfunction of a Regulator

Group A streptococcus (GAS) causes variety of diseases ranging from common pharyngitis to life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. The characteristic of invasive GAS infections has been thought to attribute to genetic changes in bacteria, however, no clear evidence has shown due to lack of an intriguingly study using serotype-matched isolates from clinical severe invasive GAS infections. In addition, rare outbreaks of invasive infections and their distinctive pathology in which infectious foci without neutrophil infiltration hypothesized us invasive GAS could evade host defense, especially neutrophil functions. Herein we report that a panel of serotype-matched GAS, which were clinically isolated from severe invasive but not from non-invaive infections, could abrogate functions of human polymorphnuclear neutrophils (PMN) in at least two independent ways; due to inducing necrosis to PMN by enhanced production of a pore-forming toxin streptolysin O (SLO) and due to impairment of PMN migration via digesting interleukin-8, a PMN attracting chemokine, by increased production of a serine protease ScpC. Expression of genes was upregulated by a loss of repressive function with the mutation of csrS gene in the all emm49 severe invasive GAS isolates. The csrS mutants from clinical severe invasive GAS isolates exhibited high mortality and disseminated infection with paucity of neutrophils, a characteristic pathology seen in human invasive GAS infection, in a mouse model. However, GAS which lack either SLO or ScpC exhibit much less mortality than the csrS-mutated parent invasive GAS isolate to the infected mice. These results suggest that the abilities of GAS to abrogate PMN functions can determine the onset and severity of invasive GAS infection

A Combination of Independent Transcriptional Regulators Shapes Bacterial Virulence Gene Expression during Infection

Transcriptional regulatory networks are fundamental to how microbes alter gene expression in response to environmental stimuli, thereby playing a critical role in bacterial pathogenesis. However, understanding how bacterial transcriptional regulatory networks function during host-pathogen interaction is limited. Recent studies in group A Streptococcus (GAS) suggested that the transcriptional regulator catabolite control protein A (CcpA) influences many of the same genes as the control of virulence (CovRS) two-component gene regulatory system. To provide new information about the CcpA and CovRS networks, we compared the CcpA and CovR transcriptomes in a serotype M1 GAS strain. The transcript levels of several of the same genes encoding virulence factors and proteins involved in basic metabolic processes were affected in both ΔccpA and ΔcovR isogenic mutant strains. Recombinant CcpA and CovR bound with high-affinity to the promoter regions of several co-regulated genes, including those encoding proteins involved in carbohydrate and amino acid metabolism. Compared to the wild-type parental strain, ΔccpA and ΔcovRΔccpA isogenic mutant strains were significantly less virulent in a mouse myositis model. Inactivation of CcpA and CovR alone and in combination led to significant alterations in the transcript levels of several key GAS virulence factor encoding genes during infection. Importantly, the transcript level alterations in the ΔccpA and ΔcovRΔccpA isogenic mutant strains observed during infection were distinct from those occurring during growth in laboratory medium. These data provide new knowledge regarding the molecular mechanisms by which pathogenic bacteria respond to environmental signals to regulate virulence factor production and basic metabolic processes during infection

Using role-play to improve students’ confidence and perceptions of communication in a simulated volcanic crisis

Traditional teaching of volcanic science typically emphasises scientific principles and tends to omit the key roles, responsibilities, protocols, and communication needs that accompany volcanic crises. This chapter provides a foundation in instructional communication, education, and risk and crisis communication research that identifies the need for authentic challenges in higher education to challenge learners and provide opportunities to practice crisis communication in real-time. We present an authentic, immersive role-play called the Volcanic Hazards Simulation that is an example of a teaching resource designed to match professional competencies. The role-play engages students in volcanic crisis concepts while simultaneously improving their confidence and perceptions of communicating science. During the role-play, students assume authentic roles and responsibilities of professionals and communicate through interdisciplinary team discussions, media releases, and press conferences. We characterised and measured the students’ confidence and perceptions of volcanic crisis communication using a mixed methods research design to determine if the role-play was effective at improving these qualities. Results showed that there was a statistically significant improvement in both communication confidence and perceptions of science communication. The exercise was most effective in transforming low-confidence and low-perception students, with some negative changes measured for our higher-learners. Additionally, students reported a comprehensive and diverse set of best practices but focussed primarily on the mechanics of science communication delivery. This curriculum is a successful example of how to improve students’ communication confidence and perceptions