Modulation of the Bacillus anthracis Secretome by the Immune Inhibitor A1 Protease

Bacteria sustain an infection by acquiring nutrients from the host to support replication. The host sequesters these nutrients as a growth-restricting strategy, a concept termed "nutritional immunity." Historically, the study of nutritional immunity has centered on iron uptake because many bacteria target hemoglobin, an abundant circulating protein, as an iron source. Left unresolved are the mechanisms that bacteria use to attain other nutrients from host sources, including amino acids. We employed a novel medium designed to mimic the chemical composition of human serum, and we show here that Bacillus anthracis, the causative agent of anthrax disease, proteolyzes human hemoglobin to liberate essential amino acids which enhance its growth. This property can be traced to the actions of InhA1, a secreted metalloprotease, and extends to at least three other serum proteins, including serum albumin. The results suggest that we must also consider proteolysis of key host proteins to be a way for bacterial pathogens to attain essential nutrients, and we provide an experimental framework to determine the host and bacterial factors involved in this process. IMPORTANCEThe mechanisms by which bacterial pathogens acquire nutrients during infection are poorly understood. Here we used a novel defined medium that approximates the chemical composition of human blood serum, blood serum mimic (BSM), to better model the nutritional environment that pathogens encounter during bacteremia. Removing essential amino acids from BSM revealed that two of the most abundant proteins in blood-hemoglobin and serum albumin-can satiate the amino acid requirement for Bacillus anthracis, the causative agent of anthrax. We further demonstrate that hemoglobin is proteolyzed by the secreted protease InhA1. These studies highlight that common blood proteins can be a nutrient source for bacteria. They also challenge the historical view that hemoglobin is solely an iron source for bacterial pathogens. There are three broad, necessary, and common elements inherent in most if not all bacterial infections of vertebrate hosts. They are (i) the initial entry of the bacterial invader and adherence to host tissues, (ii) the production of virulence factors or toxins (usually proteins) that subvert host innate and acquired immune defense mechanisms, and (iii) the acquisition and construction of biomolecules to drive cellular metabolism for the production of progeny. There has been intense investigation, and knowledge of the biochemistry, cell biology, and contribution to disease of a diverse array of bacterial adhesins and virulence factors (points i and ii identified above) is growing. However, our knowledge of nutrient acquisition during pathogenesis has lagged, perhaps because it is viewed as being a necessary and somewhat mundane process that may lack the elegant sophistication observed with virulence factors. Considering that all systemic infections are absolutely dependent on the replication of bacterial cells, identifying the mechanisms by which ba...

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“…B. anthracis UM23C1-2 (pXO1 negative [pXO1 Ϫ ], pXO2 Ϫ ) secA calY null was used to purify InhA1 (14). E. coli TG-1 was the host for cloning.…”

Section: Blood Serum Mimic (Bsm)mentioning

confidence: 99%

“…InhA1 was purified using a previously described method (14). InhA1 was expressed in B. anthracis strain UT357 cultured in LB medium for 16 h at 37°C.…”

Section: Blood Serum Mimic (Bsm)mentioning

confidence: 99%

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

et al. 2015

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“…The resulting vegetative cells spread in blood and other tissues as the causative agent of meningitis and ultimately causes death. Literature showed that among the secreted metalloproteases of B. anthracis, immune inhibitor A1 (InhA1) was found to be the single pathogenic member and during the infection it helps to cleave mammalian cell matter with addition to the modulation of the B. anthracis secreted proteins (Pflughoeft et al, 2014) and increases the permeability of blood-brain barrier resulting cerebral hemorrhages (Dhritiman et al, 2011). Previous investigation of Pflughoeft (2010) suggested that the protease cascade regulated the organism response in altering environments like reacting to a changing signal or in presence of different types of tissue.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Bacillus anthracis proteases through protein-protein interaction: an in silico study of anthrax pathogenicity

Banerjee¹,

Pal²,

Paul³

et al. 2014

TANG [HUMANITAS MEDICINE]

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Anthrax is the deadly disease for human being caused by Bacillus anthracis. Instantaneous research work on the mode of infection of the organism revealed that different proteases are involved in different steps of pathogenesis. Present study reports the in silico characterization and the detection of pathogenic proteases involved in anthrax infection through protein-protein interaction. A total of 13 acid, 9 neutral, and 1 alkaline protease of Bacillus anthracis were selected for analysing the physicochemical parameter, the protein superfamily and family search, multiple sequence alignment, phylogenetic tree construction, protein-protein interactions and motif finding. Among the 13 acid proteases, 10 were found as extracellular enzymes that interact with immune inhibitor A (InhA) and help the organism to cross the blood brain barrier during the process of infection. Multiple sequence alignment of above acid proteases revealed the position 368, 489, and 498-contained 100% conserved amino acids which could be used to deactivate the protease. Among the groups analyzed, only acid protease were found to interact with InhA, which indicated that metalloproteases of acid protease group have the capability to develop pathogenesis during B. anthracis infection. Deactivation of conserved amino acid position of germination protease can stop the sporulation and germination of B anthracis cell. The detailed interaction study of neutral and alkaline proteases could also be helpful to design the interaction network for the better understanding of anthrax disease.

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“…The two proteases that have been the most studied, Npr599 and InhA, are metalloproteases that degrade extracellular matrix proteins, syndecan-1, and several proteins involved with fibrinolysis [107]. Interestingly, the secreted proteases InhA and HtrA also appear to regulate secreted B. anthracis proteins including the adhesin BslA [108][109][110]. B.…”

Section: The Vegetative Bacillimentioning

confidence: 99%

“…anthracis has several minor virulence factors that are thought to contribute to infection in addition to the exotoxin and capsule. These include proteases and cholesterol dependent cytolysins, which degrade host proteins or lyse immune cells, as well as other factors that reduce the inflammatory response to the bacteria [105][106][107][108][109][110][111][112][113][114][118][119][120]. One such immune modulating protein is the extracellular adenosine synthase A (AdsA) [121].…”

Section: Introductionmentioning

confidence: 99%

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

Lowe

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Bacillus anthracis is a Gram positive sporulating bacterium that is the causative agent of anthrax. Early steps in dissemination are understudied due to the asymptomatic and asynchronous nature of B. anthracis infections. The goals of these studies are to establish a model of the early steps of B. anthracis infections and examine how the initial site of infection and exotoxin production affect bacterial dissemination. In these studies, a mouse model was used in combination with a bioluminescent signature tagged library to determine how B.anthracis disseminates through the host and the roles of an exotoxin component, lethal factor, in colonization and dissemination. This library allowed for real time observation of dissemination and analysis of bacterial population dynamics. In the first set of experiments, mice were infected via inhalational or subcutaneous routes with the tagged library to determine how the bacterial population changed during dissemination. In inhalational and subcutaneous infections, the disseminated population was comprised of a small subset of the original library.This indicated the presence of a population bottleneck, a random decrease in genetic diversity during the infection. Surprisingly, the diminishment and location of the bottleneck varied depending on the initial site of infection. This suggested B. anthracis exploits multiple host environments and some sites may be more permissive for dissemination than others. In the second set of experiments, mice were infected with a signature tagged library where a portion of clones lacked lethal factor. Lethal factor is known to be an important virulence factor as deletion results in attenuated or complete loss of virulence in most animal models. Host survival increased when < 10% of the library produced lethal factor, but few other differences were observed. Furthermore, decreases in the proportion of lethal factor producing clones led to fewer clones disseminating. These findings suggest that a threshold of lethal factor must be ii produced for colonization and dissemination to occur in vivo. In total, this work provides an understanding of how B. anthracis is able to rapidly disseminate from a several tissues and gives insights in where future therapeutics should be focused to reduce disease incidence.iii

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Modulation of the Bacillus anthracis Secretome by the Immune Inhibitor A1 Protease

Cited by 32 publications

References 59 publications

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

Bacillus anthracis Overcomes an Amino Acid Auxotrophy by Cleaving Host Serum Proteins

Characterization of Bacillus anthracis proteases through protein-protein interaction: an in silico study of anthrax pathogenicity

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

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