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1.
Am J Pathol ; 184(12): 3205-16, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25285720

ABSTRACT

Inhalational anthrax is caused by inhalation of Bacillus anthracis spores. The ability of B. anthracis to cause anthrax is attributed to the plasmid-encoded A/B-type toxins, edema toxin (edema factor and protective antigen) and lethal toxin (lethal factor and protective antigen), and a poly-d-glutamic acid capsule. To better understand the contribution of these toxins to the disease pathophysiology in vivo, we used B. anthracis Ames strain and isogenic toxin deletion mutants derived from the Ames strain to examine the role of lethal toxin and edema toxin after pulmonary spore challenge of cynomolgus macaques. Lethal toxin, but not edema toxin, was required to induce sustained bacteremia and death after pulmonary challenge with spores delivered via bronchoscopy. After intravenous challenge with bacilli to model the systemic phase of infection, lethal toxin contributed to bacterial proliferation and subsequent host death to a greater extent than edema toxin. Deletion of protective antigen resulted in greater loss of virulence after intravenous challenge with bacilli than deletion of lethal toxin or edema toxin alone. These findings are consistent with the ability of anti-protective antigen antibodies to prevent anthrax and suggest that lethal factor is the dominant toxin that contributes to the escape of significant numbers of bacilli from the thoracic cavity to cause anthrax after inhalation challenge with spores.


Subject(s)
Anthrax/microbiology , Antigens, Bacterial/metabolism , Bacillus anthracis/pathogenicity , Bacterial Toxins/metabolism , Lung/microbiology , Respiratory Tract Infections/microbiology , Animals , Antibodies, Bacterial/blood , Female , Macaca , Male , Spores, Bacterial/pathogenicity , Virulence , Virulence Factors/metabolism
2.
Infect Immun ; 80(7): 2414-25, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22526673

ABSTRACT

The development of therapeutics against biothreats requires that we understand the pathogenesis of the disease in relevant animal models. The rabbit model of inhalational anthrax is an important tool in the assessment of potential therapeutics against Bacillus anthracis. We investigated the roles of B. anthracis capsule and toxins in the pathogenesis of inhalational anthrax in rabbits by comparing infection with the Ames strain versus isogenic mutants with deletions of the genes for the capsule operon (capBCADE), lethal factor (lef), edema factor (cya), or protective antigen (pagA). The absence of capsule or protective antigen (PA) resulted in complete avirulence, while the presence of either edema toxin or lethal toxin plus capsule resulted in lethality. The absence of toxin did not influence the ability of B. anthracis to traffic to draining lymph nodes, but systemic dissemination required the presence of at least one of the toxins. Histopathology studies demonstrated minimal differences among lethal wild-type and single toxin mutant strains. When rabbits were coinfected with the Ames strain and the PA- mutant strain, the toxin produced by the Ames strain was not able to promote dissemination of the PA- mutant, suggesting that toxigenic action occurs in close proximity to secreting bacteria. Taken together, these findings suggest that a major role for toxins in the pathogenesis of anthrax is to enable the organism to overcome innate host effector mechanisms locally and that much of the damage during the later stages of infection is due to the interactions of the host with the massive bacterial burden.


Subject(s)
Anthrax/microbiology , Anthrax/pathology , Antigens, Bacterial/biosynthesis , Bacillus anthracis/pathogenicity , Bacterial Toxins/biosynthesis , Virulence Factors/biosynthesis , Animals , Anthrax/mortality , Antigens, Bacterial/genetics , Bacterial Capsules/genetics , Bacterial Toxins/genetics , Disease Models, Animal , Female , Gene Deletion , Histocytochemistry , Rabbits , Survival Analysis , Virulence
3.
Infect Immun ; 77(1): 429-35, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18981254

ABSTRACT

Bacillus anthracis strains harboring virulence plasmid pXO1 that encodes the toxin protein protective antigen (PA), lethal factor, and edema factor and virulence plasmid pXO2 that encodes capsule biosynthetic enzymes exhibit different levels of virulence in certain animal models. In the murine model of pulmonary infection, B. anthracis virulence was capsule dependent but toxin independent. We examined the role of toxins in subcutaneous (s.c.) infections using two different genetically complete (pXO1(+) pXO2(+)) strains of B. anthracis, strains Ames and UT500. Similar to findings for the pulmonary model, toxin was not required for infection by the Ames strain, because the 50% lethal dose (LD(50)) of a PA-deficient (PA(-)) Ames mutant was identical to that of the parent Ames strain. However, PA was required for efficient s.c. infection by the UT500 strain, because the s.c. LD(50) of a UT500 PA(-) mutant was 10,000-fold higher than the LD(50) of the parent UT500 strain. This difference between the Ames strain and the UT500 strain could not be attributed to differences in spore coat properties or the rate of germination, because s.c. inoculation with the capsulated bacillus forms also required toxin synthesis by the UT500 strain to cause lethal infection. The toxin-dependent phenotype of the UT500 strain was host phagocyte dependent, because eliminating Gr-1(+) phagocytes restored virulence to the UT500 PA(-) mutant. These experiments demonstrate that the dominant virulence factors used to establish infection by B. anthracis depend on the route of inoculation and the bacterial strain.


Subject(s)
Anthrax/microbiology , Anthrax/pathology , Bacillus anthracis/pathogenicity , Virulence Factors/physiology , Animals , Antigens, Bacterial/genetics , Antigens, Bacterial/physiology , Bacterial Toxins/genetics , Female , Lethal Dose 50 , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Phagocytes/immunology , Phagocytes/microbiology , Survival Analysis , Virulence , Virulence Factors/genetics
4.
PLoS One ; 3(3): e1824, 2008 Mar 19.
Article in English | MEDLINE | ID: mdl-18350160

ABSTRACT

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.


Subject(s)
Antigens, Bacterial/immunology , Bacillus anthracis/immunology , Animals , Bacillus anthracis/genetics , Gene Expression Profiling , Macaca mulatta , Mice , Mice, Inbred BALB C , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
5.
Infect Immun ; 75(4): 1757-64, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17242059

ABSTRACT

Effective treatment of anthrax is hampered by our limited understanding of the pathophysiology of Bacillus anthracis infection. We used a genetically complete (pXO1(+) pXO2(+)) virulent B. anthracis strain and four isogenic toxin-null mutants to determine the effects of the anthrax edema toxin (ET; edema factor [EF] plus protective antigen [PA]) and lethal toxin (LT; lethal factor [LF] plus PA) on the host innate response during systemic infection. Using the spleen as an indicator for host response, we found that intravenous inoculation of LT-deficient mutants into C57BL/6 mice significantly increased production of several cytokines over that observed after infection with the parent strain or an EF-deficient mutant. Bacteria producing one or both of the toxins were capable of inducing significant apoptosis of cells present in spleens, whereas apoptosis was greatly reduced in mice infected with nontoxigenic mutants. Mice infected with toxin-producing strains also showed increased splenic neutrophil recruitment compared to mice infected with nontoxigenic strains and neutrophil depletion prior to infection with toxin-producing strains, leading to decreased levels of apoptosis. Together, these studies indicate that anthrax LT suppresses cytokine secretion during infection, but both EF and LF play roles in inducing neutrophil recruitment and enhancing apoptosis. Interestingly, in the absence of LF the effect of EF-induced cell recruitment is further enhanced, perhaps because LF so effectively suppresses the secretion of chemokines.


Subject(s)
Anthrax/immunology , Antigens, Bacterial/biosynthesis , Bacillus anthracis/immunology , Bacterial Toxins/biosynthesis , Animals , Anthrax/microbiology , Anthrax/pathology , Antigens, Bacterial/genetics , Apoptosis , Bacillus anthracis/pathogenicity , Bacterial Toxins/genetics , Cell Survival , Colony Count, Microbial , Cytokines/biosynthesis , Disease Models, Animal , Female , Immunity, Innate , In Situ Nick-End Labeling , Leukocyte Reduction Procedures , Mice , Mice, Inbred C57BL , Neutrophils/immunology , Spleen/immunology , Spleen/microbiology , Spleen/pathology
6.
Infect Immun ; 74(11): 6067-74, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16923785

ABSTRACT

Bacillus anthracis, the etiologic agent of anthrax, produces at least three primary virulence factors: lethal toxin, edema toxin, and a capsule. The capsule is absolutely required for dissemination and lethality in a murine model of inhalation anthrax, yet the roles for the toxins during infection are ill-defined. We show in a murine model that when spores of specific toxin-null mutants are introduced into the lung, dissemination and lethality are comparable to those of the parent strain. Mutants lacking one or more of the structural genes for the toxin proteins, i.e., protective antigen, lethal factor, and edema factor, disseminated from the lung to the spleen at rates similar to that of the virulent parental strain. The 50% lethal dose (LD50) and mean time to death (MTD) of the mutants did not differ significantly from those of the parent. The LD50s or MTDs were also unaffected relative to those of the parent strain when mice were inoculated intravenously with vegetative cells. Nonetheless, histopathological examination of tissues revealed subtle but distinct differences in infections by the parent compared to some toxin mutants, suggesting that the host response is affected by toxin proteins synthesized during infection.


Subject(s)
Anthrax/microbiology , Anthrax/mortality , Antigens, Bacterial/genetics , Bacillus anthracis/genetics , Bacillus anthracis/pathogenicity , Bacterial Toxins/genetics , Genes, Lethal , Pneumonia, Bacterial/microbiology , Pneumonia, Bacterial/mortality , Animals , Anthrax/pathology , Antigens, Bacterial/biosynthesis , Bacterial Capsules/biosynthesis , Bacterial Capsules/genetics , Bacterial Toxins/biosynthesis , Disease Models, Animal , Female , Mice , Mice, Inbred BALB C , Pneumonia, Bacterial/pathology , Spores, Bacterial/genetics , Virulence Factors/genetics
7.
J Bacteriol ; 187(15): 5108-14, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16030203

ABSTRACT

The poly-d-glutamic acid capsule of Bacillus anthracis is essential for virulence. Control of capsule synthesis occurs at the level of transcription and involves positive regulation of the capsule biosynthetic operon capBCAD by a CO2/bicarbonate signal and three plasmid-borne regulators: atxA, acpA, and acpB. Although the molecular mechanism for control of cap transcription is unknown, atxA affects cap expression via positive control of acpA and acpB, two genes with partial functional similarity. Transcriptional analyses of a genetically complete strain indicate that capB expression is several hundred-fold higher during growth in 5% CO2 compared to growth in air. atxA was expressed appreciably during growth in air and induced only 2.5-fold by CO2. In contrast, expression of acpA and acpB was induced up to 23-fold and 59-fold, respectively, by CO2. The 5'-end mapping of gene transcripts revealed atxA-regulated and atxA-independent apparent transcription start sites for capB, acpA, and acpB. Transcripts mapping to all atxA-regulated start sites were increased during growth in elevated CO2. The acpA gene has one atxA-regulated and one atxA-independent start site. acpB lies downstream of capBCAD. A single atxA-independent start site maps immediately upstream of acpB. atxA-mediated control of acpB appears to occur via transcriptional read-through from atxA-dependent start sites 5' of capB. One atxA-independent and two atxA-regulated start sites map upstream of capB. Transcription from the atxA-regulated start sites of capBCAD was reduced significantly in an acpA acpB double mutant but unaffected in mutants with deletion of only acpA or acpB, in agreement with the current model for epistatic relationships between the regulators.


Subject(s)
Bacillus anthracis/genetics , Bacterial Capsules/genetics , RNA, Messenger/genetics , Bacillus anthracis/growth & development , Bacillus anthracis/metabolism , Bacterial Capsules/biosynthesis , Bacterial Proteins/genetics , Base Sequence , Bicarbonates , Carbon Dioxide , Molecular Sequence Data , Operon , Trans-Activators/genetics
8.
EMBO J ; 24(1): 221-7, 2005 Jan 12.
Article in English | MEDLINE | ID: mdl-15616593

ABSTRACT

Bacillus anthracis, the agent of anthrax, produces a poly-D-glutamic acid capsule that has been implicated in virulence. Many strains missing pXO2 (96 kb), which harbors the capsule biosynthetic operon capBCAD, but carrying pXO1 (182 kb) that harbors the anthrax toxin genes, are attenuated in animal models. Also, noncapsulated strains are readily phagocytosed by macrophage cell lines, whereas capsulated strains are resistant to phagocytosis. We show that a strain carrying both virulence plasmids but deleted specifically for capBCAD is highly attenuated in a mouse model for inhalation anthrax. The parent strain and capsule mutant initiated germination in the lungs, but the capsule mutant did not disseminate to the spleen. A mutant harboring capBCAD but deleted for the cap regulators acpA and acpB was also significantly attenuated, in agreement with the capsule-negative phenotype during in vitro growth. Surprisingly, an acpB mutant, but not an acpA mutant, displayed an elevated LD(50) and reduced ability to disseminate, indicating that acpA and acpB are not true functional homologs and that acpB may play a larger role in virulence than originally suspected.


Subject(s)
Anthrax/microbiology , Bacillus anthracis/metabolism , Bacillus anthracis/pathogenicity , Bacterial Capsules/biosynthesis , Bacterial Capsules/genetics , Animals , Bacillus anthracis/genetics , Disease Models, Animal , Female , Gene Expression Regulation, Bacterial , Lung/microbiology , Lung/pathology , Mice , Mice, Inbred BALB C , Operon , Spleen/microbiology , Spleen/pathology , Survival Rate , Virulence Factors/genetics , Virulence Factors/metabolism
9.
J Bacteriol ; 186(2): 307-15, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14702298

ABSTRACT

Two regulatory genes, acpA and atxA, have been reported to control expression of the Bacillus anthracis capsule biosynthesis operon capBCAD. The atxA gene is located on the virulence plasmid pXO1, while pXO2 carries acpA and the cap genes. acpA has been viewed as the major regulator of the cap operon because it is essential for capsule gene expression in a pXO1(-) pXO2(+) strain. atxA is essential for toxin gene transcription but has also been implicated in control of the cap genes. The molecular functions of the regulatory proteins are unknown. We examined cap gene expression in a genetically complete pXO1(+) pXO2(+) strain. Our results indicate that another pXO2 gene, acpB (previously called pXO2-53; accession no. NC002146.1:49418-50866), has a role in cap expression. The predicted amino acid sequence of AcpB is 62% similar to that of AcpA and 50% similar to that of AtxA. Assessment of cap gene transcription revealed that cap expression was not affected in a pXO1(+) pXO2(+) acpB-null mutant and was slightly reduced in an isogenic acpA mutant. However, cap gene expression was abolished in an acpA acpB double mutant. Microscopic examination of capsule synthesis by the mutants corroborated these findings. acpA and acpB expression is controlled by atxA; capsule synthesis and transcription of acpA and acpB were markedly reduced in an atxA mutant. The data suggest that, in a strain containing both virulence plasmids, atxA is the major regulator of capsule synthesis and controls capBCAD expression indirectly, via positive regulation of acpA and acpB.


Subject(s)
Bacterial Capsules/biosynthesis , Bacterial Proteins/physiology , Trans-Activators/physiology , Amino Acid Sequence , Bacterial Capsules/genetics , Bacterial Proteins/genetics , Carbon Dioxide/pharmacology , Molecular Sequence Data , RNA, Messenger/analysis , Trans-Activators/genetics
10.
Infect Immun ; 71(5): 2736-43, 2003 May.
Article in English | MEDLINE | ID: mdl-12704148

ABSTRACT

Control of anthrax toxin and capsule synthesis, the two major virulence factors of Bacillus anthracis, has been associated with two regulatory genes, atxA and acpA, located on virulence plasmids pXO1 and pXO2, respectively. We used transcriptional profiling to determine whether atxA and/or acpA control genes other than those already described and to investigate functional similarities of the regulators. Transcription was assessed in a pXO1(+) pXO2(+) parent strain and in isogenic mutants in which one or both regulatory genes were deleted. We determined that in addition to the toxin and capsule genes, atxA controls expression of numerous other genes on both plasmids and the chromosome. Generally, plasmid-encoded genes were more highly regulated than chromosomal genes, and both positive and negative effects were observed. Certain atxA-regulated genes were affected synergistically in an atxA acpA mutant. Yet overall, acpA appears to be a minor regulator with fewer targets than atxA. In contrast to previous reports of acpA function in attenuated strains, acpA had a minimal influence on capsule gene transcription and capsule synthesis in a genetically complete strain. Surprisingly, acpA expression was positively affected by atxA, although atxA-activated capsule gene transcription is not acpA dependent. The newly discovered atxA-regulated targets include genes predicted to encode secreted proteins and proteins with roles in transcriptional regulation and signaling. Regulation of chromosomal genes by atxA is particularly intriguing, given that many of the target genes have homologues in other Bacillus species that lack atxA homologues. Given the global effect of atxA on gene expression in B. anthracis, previous assumptions regarding reduced virulence of strains harboring single plasmids must be reassessed and the potential roles of newly identified atxA-regulated genes should be investigated.


Subject(s)
Bacillus anthracis/genetics , Bacillus anthracis/pathogenicity , Bacterial Proteins/genetics , Genes, Regulator/physiology , Plasmids , Trans-Activators/genetics , Bacterial Capsules/physiology , Bacterial Proteins/physiology , Gene Expression Regulation, Bacterial , Open Reading Frames , Trans-Activators/physiology , Virulence
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