Functional Analysis of luxS in Staphylococcus aureus Reveals a Role in Metabolism but Not Quorum Sensing

ABSTRACT The function of AI-2 in many bacteria and the physiological role of LuxS, the enzyme responsible for its production, remain matters of debate. Here, we show that in Staphylococcus aureus the luxS gene forms a monocistronic transcriptional unit under the control of a σ70-dependent promoter. The gene was transcribed throughout growth under a variety of conditions, including intracellular growth in MAC-T cells. AI-2 was produced in rich media under aerobic and anaerobic conditions, peaking during the transition to stationary phase, but was hardly detectable in a sulfur-limited defined medium. In the presence of glucose or under anaerobic conditions, cultures retained considerable AI-2 activity after entry into stationary phase. Inactivation of luxS in various S. aureus strains did not affect virulence-associated traits, such as production of hemolysins and extracellular proteases, biofilm formation, and the agr signaling system. Conversely, AI-2 production remained unchanged in an agr mutant. However, luxS mutants grown in a sulfur-limited defined medium exhibited a growth defect. When grown together with the wild type in mixed culture, luxS mutants of various S. aureus strains showed reduced ability to compete for growth under these conditions. In contrast, a complemented luxS mutant grew as well as the parent strain, suggesting that the observed growth defect was of an intracellular nature and had not been caused by either second-site mutations or the lack of a diffusible factor. However, the LuxS/AI-2 system does not appear to contribute to the overall fitness of S. aureus RN6390B during intracellular growth in epithelial cells: the wild type and a luxS mutant showed very similar growth patterns after their internalization by MAC-T cells.

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Construction and Characterization of aMycobacterium tuberculosis Mutant Lacking the Alternate Sigma Factor Gene, sigF

Infection and Immunity ◽

10.1128/iai.68.10.5575-5580.2000 ◽

2000 ◽

Vol 68 (10) ◽

pp. 5575-5580 ◽

Cited By ~ 99

Author(s):

Ping Chen ◽

Rafael E. Ruiz ◽

Qing Li ◽

Richard F. Silver ◽

William R. Bishai

Keyword(s):

Stationary Phase ◽

Sigma Factor ◽

Type Strain ◽

Wild Type Strain ◽

Axenic Culture ◽

Wild Type ◽

Intracellular Growth ◽

Factor Gene ◽

Time To Death

ABSTRACT The alternate RNA polymerase sigma factor gene, sigF, which is expressed in stationary phase and under stress conditions in vitro, has been deleted in the virulent CDC1551 strain ofMycobacterium tuberculosis. The growth rate of the ΔsigF mutant was identical to that of the isogenic wild-type strain in exponential phase, although in stationary phase the mutant achieved a higher density than the wild type. The mutant showed increased susceptibility to rifampin and rifapentine. Additionally, the ΔsigF mutant displayed diminished uptake of chenodeoxycholate, and this effect was reversed by complementation with a wild-type sigF gene. No differences in short-term intracellular growth between mutant and wild-type organisms within human monocytes were observed. Similarly, the organisms did not differ in their susceptibilities to lymphocyte-mediated inhibition of intracellular growth. However, mice infected with the ΔsigF mutant showed a median time to death of 246 days compared with 161 days for wild-type strain-infected animals (P < 0.001). These data indicate that M. tuberculosis sigF is a nonessential alternate sigma factor both in axenic culture and for survival in macrophages in vitro. While the ΔsigF mutant produces a lethal infection of mice, it is less virulent than its wild-type counterpart by time-to-death analysis.

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The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

Microbiology ◽

10.1099/mic.0.27563-0 ◽

2005 ◽

Vol 151 (1) ◽

pp. 167-182 ◽

Cited By ~ 36

Author(s):

Urs Albers ◽

Katrin Reus ◽

Howard A. Shuman ◽

Hubert Hilbi

Keyword(s):

Legionella Pneumophila ◽

Lipid A ◽

Sequence Similarity ◽

Acanthamoeba Castellanii ◽

Growth Defect ◽

Wild Type ◽

Intracellular Growth ◽

Plate Test ◽

Raw264.7 Macrophages ◽

Mutant Strains

Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

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Characterization of a Legionella pneumophila relA Insertion Mutant and Roles of RelA and RpoS in Virulence Gene Expression

Journal of Bacteriology ◽

10.1128/jb.184.1.67-75.2002 ◽

2002 ◽

Vol 184 (1) ◽

pp. 67-75 ◽

Cited By ~ 83

Author(s):

Tal Zusman ◽

Ohad Gal-Mor ◽

Gil Segal

Keyword(s):

Gene Expression ◽

Stationary Phase ◽

Gene Product ◽

Legionella Pneumophila ◽

Type Strain ◽

Wild Type Strain ◽

Insertion Mutant ◽

Wild Type ◽

Intracellular Growth ◽

Virulence Gene Expression

ABSTRACT To investigate the involvement of RelA in the regulation of Legionella pneumophila virulence, a deletion substitution was constructed in the relA gene. The relA knockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when the relA gene product was supplied on a plasmid. The effect of the relA mutation was examined with two systems that are known to be expressed during the stationary phase in L. pneumophila. Pigment production was found to be dependent on the relA gene product, and only one-half as much pigment was produced by the relA mutant as by the wild-type strain. Flagellum gene expression was also found to be dependent on the relA gene product, as determined with a flaA::lacZ fusion. However, the relA gene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii. To determine the involvement of the relA gene product in expression of L. pneumophila genes required for intracellular growth (icm/dot genes), nine icm::lacZ fusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression in relA mutant strains. Expression of only one of the icm::lacZ fusions was moderately reduced in the relA mutant strain. Expression of the nine icm::lacZ fusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained. We concluded that RelA is dispensable for intracellular growth of L. pneumophila in the two hosts examined and that both RelA and RpoS play minor roles in L. pneumophila icm/dot gene expression.

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Lactate Acquisition Promotes Successful Colonization of the Murine Genital Tract by Neisseria gonorrhoeae

Infection and Immunity ◽

10.1128/iai.01530-06 ◽

2006 ◽

Vol 75 (3) ◽

pp. 1318-1324 ◽

Cited By ~ 34

Author(s):

Rachel M. Exley ◽

Hong Wu ◽

Jonathan Shaw ◽

Muriel C. Schneider ◽

Harry Smith ◽

...

Keyword(s):

Carbon Source ◽

Tract Infection ◽

Neisseria Gonorrhoeae ◽

Genital Tract ◽

Defined Medium ◽

Growth Defect ◽

Wild Type ◽

Genital Tract Infection ◽

Lactate Utilization

ABSTRACT Previous studies on Neisseria gonorrhoeae have demonstrated that metabolism of lactate in the presence of glucose increases the growth rate of the bacterium and enhances its resistance to complement-mediated killing. Although these findings in vitro suggest that the acquisition of lactate promotes gonococcal colonization, the significance of this carbon source to the survival of the gonococcus in vivo remains unknown. To investigate the importance of lactate utilization during Neisseria gonorrhoeae genital tract infection, we identified the gene lctP, which encodes the gonococcal lactate permease. A mutant that lacks a functional copy of lctP was unable to take up exogenous lactate and did not grow in defined medium with lactate as the sole carbon source, in contrast to the wild-type and complemented strains; the mutant strain exhibited no growth defect in defined medium containing glucose. In defined medium containing physiological concentrations of lactate and glucose, the lctP mutant demonstrated reduced early growth and increased sensitivity to complement-mediated killing compared with the wild-type strain; the enhanced susceptibility to complement was associated with a reduction in lipopolysaccharide sialylation of the lctP mutant. The importance of lactate utilization during colonization was evaluated in the murine model of lower genital tract infection. The lctP mutant was significantly attenuated in its ability to colonize and survive in the genital tract, while the complemented mutant exhibited no defect for colonization. Lactate is a micronutrient in the genital tract that contributes to the survival of the gonococcus.

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The PmrA/PmrB Two-Component System of Legionella pneumophila Is a Global Regulator Required for Intracellular Replication within Macrophages and Protozoa

Infection and Immunity ◽

10.1128/iai.01081-08 ◽

2008 ◽

Vol 77 (1) ◽

pp. 374-386 ◽

Cited By ~ 50

Author(s):

Souhaila Al-Khodor ◽

Sergey Kalachikov ◽

Irina Morozova ◽

Christopher T. Price ◽

Yousef Abu Kwaik

Keyword(s):

Legionella Pneumophila ◽

Wild Type Strain ◽

Growth Defect ◽

Wild Type ◽

Intracellular Growth ◽

Global Effect ◽

Two Component System ◽

Human Macrophages ◽

Two Component ◽

Intracellular Proliferation

ABSTRACT To examine the role of the PmrA/PmrB two-component system (TCS) of Legionella pneumophila in global gene regulation and in intracellular infection, we constructed pmrA and pmrB isogenic mutants by allelic exchange. Genome-wide microarray gene expression analyses of the pmrA and pmrB mutants at both the exponential and the postexponential phases have shown that the PmrA/PmrB TCS has a global effect on the expression of 279 genes classified into nine groups of genes encoding eukaryotic-like proteins, Dot/Icm apparatus and secreted effectors, type II-secreted proteins, regulators of the postexponential phase, stress response genes, flagellar biosynthesis genes, metabolic genes, and genes of unknown function. Forty-one genes were differentially regulated in the pmrA or pmrB mutant, suggesting a possible cross talk with other TCSs. The pmrB mutant is more sensitive to low pH than the pmrA mutant and the wild-type strain, suggesting that acidity may trigger this TCS. The pmrB mutant exhibits a significant defect in intracellular proliferation within human macrophages, Acanthamoeba polyphaga, and the ciliate Tetrahymena pyriformis. In contrast, the pmrA mutant is defective only in the ciliate. Despite the intracellular growth defect within human macrophages, phagosomes harboring the pmrB mutant exclude late endosomal and lysosomal markers and are remodeled by the rough endoplasmic reticulum. Similar to the dot/icm mutants, the intracellular growth defect of the pmrB mutant is totally rescued in cis within communal phagosomes harboring the wild-type strain. We conclude that the PmrA/PmrB TCS has a global effect on gene expression and is required for the intracellular proliferation of L. pneumophila within human macrophages and protozoa. Differences in gene regulation and intracellular growth phenotypes between the pmrA and pmrB mutant suggests a cross talk with other TCSs.

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Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation inStaphylococcus aureusbut dispensable for viability in anaerobic conditions

10.1101/216887 ◽

2017 ◽

Author(s):

Merve S. Zeden ◽

Christopher F. Schuster ◽

Lisa Bowman ◽

Qiyun Zhong ◽

Huw D. Williams ◽

...

Keyword(s):

Amino Acid ◽

Adenosine Monophosphate ◽

Defined Medium ◽

Growth Defect ◽

Uptake System ◽

Osmotic Regulation ◽

Bacterial Cells ◽

Rich Medium ◽

Anaerobic Conditions

ABSTRACTCyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such asStaphylococcus aureus. However, the exact role of this molecule has not been identified.dacA, theS. aureusgene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that anS. aureus dacAmutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality ofdacAwere identified inalsTandopuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system inS. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained inhepS, hemB, ctaAandqoxB, coding for proteins required for respiration. Furthermore, we show thatdacAis dispensable for growth in anaerobic conditions. Together, these finding reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration inS. aureus.

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The Mycobacterium tuberculosis SecA2 System Subverts Phagosome Maturation To Promote Growth in Macrophages

Infection and Immunity ◽

10.1128/iai.05987-11 ◽

2012 ◽

Vol 80 (3) ◽

pp. 996-1006 ◽

Cited By ~ 51

Author(s):

Jonathan Tabb Sullivan ◽

Ellen F. Young ◽

Jessica R. McCann ◽

Miriam Braunstein

Keyword(s):

Mycobacterium Tuberculosis ◽

Protein Export ◽

Growth Defect ◽

Phagosome Maturation ◽

Wild Type ◽

Intracellular Growth ◽

Maturation Arrest ◽

Content Type ◽

Late Endosomes ◽

Export System

The ability ofMycobacterium tuberculosisto grow in macrophages is critical to the virulence of this important pathogen. One wayM. tuberculosisis thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest byM. tuberculosisis not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest forM. tuberculosisgrowth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. InM. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ΔsecA2mutant ofM. tuberculosiswere more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-typeM. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ΔsecA2mutant, which demonstrated that the phagosome maturation arrest defect of the ΔsecA2mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest forM. tuberculosisgrowth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.

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Disruption of Phosphate Homeostasis Sensitizes Staphylococcus aureus to Nutritional Immunity

Infection and Immunity ◽

10.1128/iai.00102-20 ◽

2020 ◽

Vol 88 (6) ◽

Cited By ~ 1

Author(s):

Jessica L. Kelliher ◽

Erin B. Brazel ◽

Jana N. Radin ◽

Eliot S. Joya ◽

Paola K. Párraga Solórzano ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Defined Medium ◽

Wild Type ◽

Two Component System ◽

Phosphate Homeostasis ◽

Component System ◽

Content Type ◽

Nutritional Immunity ◽

Two Component ◽

Overexpressing Strain

ABSTRACT To control infection, mammals actively withhold essential nutrients, including the transition metal manganese, by a process termed nutritional immunity. A critical component of this host response is the manganese-chelating protein calprotectin. While many bacterial mechanisms for overcoming nutritional immunity have been identified, the intersection between metal starvation and other essential inorganic nutrients has not been investigated. Here, we report that overexpression of an operon encoding a highly conserved inorganic phosphate importer, PstSCAB, increases the sensitivity of Staphylococcus aureus to calprotectin-mediated manganese sequestration. Further analysis revealed that overexpression of pstSCAB does not disrupt manganese acquisition or result in overaccumulation of phosphate by S. aureus. However, it does reduce the ability of S. aureus to grow in phosphate-replete defined medium. Overexpression of pstSCAB does not aberrantly activate the phosphate-responsive two-component system PhoPR, nor was this two-component system required for sensitivity to manganese starvation. In a mouse model of systemic staphylococcal disease, a pstSCAB-overexpressing strain is significantly attenuated compared to wild-type S. aureus. This defect is partially reversed in a calprotectin-deficient mouse, in which manganese is more readily available. Given that expression of pstSCAB is regulated by PhoPR, these findings suggest that overactivation of PhoPR would diminish the ability of S. aureus to resist nutritional immunity and cause infection. As PhoPR is also necessary for bacterial virulence, these findings imply that phosphate homeostasis represents a critical regulatory node whose activity must be precisely controlled in order for S. aureus and other pathogens to cause infection.

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Tryptophan Prototrophy Contributes to Francisella tularensis Evasion of Gamma Interferon-Mediated Host Defense

Infection and Immunity ◽

10.1128/iai.01349-10 ◽

2011 ◽

Vol 79 (6) ◽

pp. 2356-2361 ◽

Cited By ~ 13

Author(s):

Ping Chu ◽

Annette R. Rodriguez ◽

Bernard P. Arulanandam ◽

Karl E. Klose

Keyword(s):

Francisella Tularensis ◽

Host Defense ◽

Defined Medium ◽

Gamma Interferon ◽

Wild Type ◽

Intracellular Growth ◽

Content Type ◽

Interferon Receptor ◽

High Virulence ◽

Ifn Γ

ABSTRACTFrancisella tularensisis able to survive and replicate within host macrophages, a trait that is associated with the high virulence of this bacterium. ThetrpABgenes encode the enzymes required for the final two steps in tryptophan biosynthesis, with TrpB being responsible for the conversion of indole to tryptophan. Consistent with this function, anF. tularensissubsp.novicidatrpBmutant is unable to grow in defined medium in the absence of tryptophan. ThetrpBmutant is also attenuated for virulence in a mouse pulmonary model of tularemia. However, thetrpBmutant remains virulent in gamma interferon receptor-deficient (IFN-γR−/−) mice, demonstrating that IFN-γ-mediated signaling contributes to clearance of thetrpBmutant. IFN-γ limits intracellular survival of thetrpBmutant within bone marrow-derived macrophages from wild-type but not IFN-γR−/−mice. AnF. tularensissubsp.tularensis trpBmutant is also attenuated for virulence in mice and survival within IFN-γ-treated macrophages, indicating that tryptophan prototrophy is also important in a human-virulentF. tularensissubspecies. These results demonstrate thattrpBcontributes toF. tularensisvirulence by enabling intracellular growth under IFN-γ-mediated tryptophan limitation.

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Multiple T Cell Subsets Control Francisella tularensis LVS Intracellular Growth Without Stimulation Through Macrophage Interferon γ Receptors

Journal of Experimental Medicine ◽

10.1084/jem.20030687 ◽

2003 ◽

Vol 198 (3) ◽

pp. 379-389 ◽

Cited By ~ 81

Author(s):

Siobhán C. Cowley ◽

Karen L. Elkins

Keyword(s):

T Cells ◽

T Cell ◽

Francisella Tularensis ◽

T Cell Subsets ◽

Wild Type ◽

Intracellular Growth ◽

Tnf Α ◽

Ifn Γ

A variety of data suggest that in vivo production of interferon (IFN)-γ is necessary, but not sufficient, for expression of secondary protective immunity against intracellular pathogens. To discover specific IFN-γ–independent T cell mediated mechanisms, we took advantage of an in vitro culture system that models in vivo immune responses to the intracellular bacterium Francisella tularensis live vaccine strain (LVS). LVS-immune lymphocytes specifically controlled 99% of the growth of LVS in wild-type murine bone marrow–derived macrophages. Surprisingly, LVS-immune lymphocytes also inhibited LVS intracellular growth by as much as 95% in macrophages derived from IFN-γ receptor knockout (IFNγR KO) mice. CD8+ T cells, and to a lesser degree CD4+ T cells, controlled LVS intracellular growth in both wild-type and IFNγR KO macrophages. Further, a unique population of Thy1+αβ+CD4−CD8− cells that was previously suggested to operate during secondary immunity to LVS in vivo strongly controlled LVS intracellular growth in vitro. A large proportion of the inhibition of LVS intracellular growth in IFNγR KO macrophages by all three T cell subsets could be attributed to tumor necrosis factor (TNF) α. Thus, T cell mechanisms exist that control LVS intracellular growth without acting through the IFN-γ receptor; such control is due in large part to TNF-α, and is partially mediated by a unique double negative T cell subpopulation.

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