We first constructed a bclA deletion mutant of B. anthracis Sterne strain 34F2. This deletion cleanly excised the entire bclA open reading frame, replacing it with an XmaI site (CCCGGG). Thus, this mutation was not expected to exert polarity on downstream genes. It should also be noted that although an examination of the annotated genomic sequence of B. anthracis Ames indicates the presence of additional genes downstream of, and in the same orientation as, bclA, the presence of a predicted rho-independent terminator just downstream of bclA also supports the view that this deletion does not affect the expression of downstream genes.
Next, we tested SSPEs of wild-type and 34F2-ΔbclA
spores for the presence or absence of BclA by Western blot analysis (Fig. ). We observed a broad high-molecular-weight band in the SSPE from 34F2 spores. That band (~180 kDa) was smaller but similar in size to that for glycosylated wild-type BclA (~250 kDa) reported previously by Sylvestre et al. (23
). Our E. coli
-derived rBclA control migrated at ~70 kDa, a size consistent with that noted previously by Steichen et al. (22
) for rBclA. As anticipated from the manner in which the 34F2-ΔbclA
mutant was derived, we detected no BclA band in the SSPE of 34F2-ΔbclA
We then compared the growth and sporulation capacities of wild-type 34F2 with 34F2-ΔbclA. We found that the strains grew with comparable kinetics and to indistinguishable levels (Fig. ). To evaluate the sporulation capacity of the wild-type and mutant strains, spore formation over time in broth culture was assessed by both optical density measurement at 600 nm (Fig. ) and phase-contrast microscopy of samples taken at 24-, 48-, 72-, 96-h time points (data not shown). Results by both sporulation assessment methods were similar for the wild-type and mutant strains.
We then used 34F2-ΔbclA
to address the influence of BclA on four properties of the spore: germination rate, virulence, adhesion to extracellular matrix proteins, and hydrophobicity. For the germination assay, we elected to use CFU in the presence and absence of heat as our measurement because a change from heat resistance to heat sensitivity is considered to be an irreversible step in the process of spore germination (12
). We found that the mutant spores germinated at a faster rate in vitro than did wild-type spores between 30 and 60 min of incubation in germination medium and that the mutant spores germinated to a statistically significantly greater level than the wild type at 45 and 60 min of incubation (Fig. ). Although Bozue et al. reported no statistically significant differences in germination between the Ames strain and its bclA
), that mutant appeared to germinate to a somewhat greater extent and at a slightly faster rate than the wild type, as evaluated by spectrofluorometric assays for germination.
FIG. 2. Germination curve of 34F2 and 34F2-ΔbclA spores. Germination is measured here as a loss of heat resistance. The experiment was repeated three times. Analysis of variance showed statistically significant differences (P values of 0.001 and 0.007 (more ...)
When we compared the relative LD50
s of mutant and wild-type spores by intranasal or subcutaneous routes of administration, no statistically significant differences between 34F2 and 34F2-ΔbclA
were noted (Table ). This finding is in keeping with reports described previously by Sylvestre et al. (23
), who used a Sterne-like strain, and Bozue and colleagues, who used Ames (5
). However, the LD50
values that we obtained were always lower for the mutant strain (Table ). Furthermore, the mean time to death (MTD) of mice given a 106
dose of wild-type 34F2 spores was 4.2 days for the intranasal route and 4.4 days for the subcutaneous route compared to 3 days for mice that received 34F2-ΔbclA
spores by both the intranasal and subcutaneous routes. Thus, the MTD was shorter for the 106
dose by both the subcutaneous and intranasal routes for the mutant strain (P
= 0.006). These two findings taken together indicate that 34F2-ΔbclA
spores germinate slightly better or faster in vivo (as they appear to do in vitro) or perhaps associate with host cells more avidly than do wild-type spores. Indeed, Bozue et al. reported less efficient recovery of ΔbclA
spores of the Ames strain from lungs of mice upon bronchoalveolar lavage at days 2 and 4 in an aerosolized spore challenge (5
). Those authors speculated that this reduced clearance of the mutant spores from the lungs of these animals might reflect the better binding of mutant spores than wild-type spores to host cells lining the lungs or airways. In fact, very recently, Bozue et al. (6
) demonstrated that bclA
mutant spores adhered to epithelial cell lines much better than did wild-type Ames spores. However, those investigators noted that such adhesion differences between their BclA Ames mutant strain and Ames did not extend to macrophages (6
Comparison of LD50 values of spores of B. anthracis Sterne strain 34F2 and 34F2-ΔbclA
To evaluate the possibility that BclA might modulate the surface of the spore in a manner that influences the degree of association of spores with host matrix proteins, we compared how well fibronectin and laminin bound to wild-type and bclA
mutant spores We found that the interaction of both fibronectin and laminin with 34F2-ΔbclA
spores was more extensive than that with 34F2 spores (Fig. ). These results suggest that when BclA is removed from the surface of the spores, another protein(s) may become exposed and adhere to the two extracellular matrix proteins tested. Moreover, our data on the greater stickiness of the extracellular matrix components laminin and fibronectin to BclA-negative spores is consistent with the aforementioned report by Bozue et al. (6
) on the enhanced adhesion of bclA
mutant Ames spores to epithelial cells compared to the parental strain.
FIG. 3. Comparison of adherence of matrix proteins to 34F2 and 34F2-ΔbclA spores. (A) Binding of fibronectin to spores. (B) Binding of laminin to spores. Data are shown as means ± standard deviations of values obtained from three experiments done (more ...)
One possible explanation as to why matrix proteins bound less well to BclA-positive spores than to BclA-negative spores is that BclA affects the hydrophobic nature of the spore. That bacterial spores are hydrophobic has been demonstrated for several species of Bacillus
), and, probably as a consequence, these types of spores adhere strongly to inanimate surfaces like microtiter plates (1
). Moreover, Bacillus cereus
spores bind to Caco-2 cells through surface hydrophobic interactions (2
). The possibility that the degree of hydrophobicity of spores may be linked to the presence of an exosporium was first suggested by Takubo et al. (24
), who showed that Bacillus megaterium
QMB1551 spores with a defective or absent exosporium exhibit reduced affinity for hexadecane. Moreover, spores of Bacillus subtilis
, B. licheniformis
, and B. macerans
do not have distinct exosporium layers and are less hydrophobic than are exosporium-containing B. cereus
, B. brevis
, and B. thuringiensis
). Furthermore, a reduction in partitioning to hexadecane was successfully used by Bailey-Smith et al. to enrich for exosporium mutants of B. cereus
). Thus, several studies indicate that the surface hydrophobicity of certain spores is attributable to the presence of an exosporium. However, the specific structural component(s) that contributes to the hydrophobic nature of the spore is not known.
To test our theory about the impact of BclA on the hydrophobicity of spores, we evaluated the percent hydrophobicity of spores at various molarities of hexadecane. We found that the hydrophobicity of wild-type spores was greater than that of ΔbclA spores at each of the four hexadecane concentrations selected (Fig. ). We confirmed the findings of this BATH assay with a second hydrocarbon, toluene, at 0.1 ml per ml of spore suspension (853 μM toluene). Again, the wild-type spores showed higher hydrophobicity with toluene than did mutant spores (Fig. ). The 34F2 spores were more hydrophobic by HIC than were 34F2-ΔbclA spores (data not shown), although the absolute hydrophobicities of the spores as measured by the BATH and HIC techniques did not agree.
FIG. 4. Comparison of hydrophobicity of 34F2 and 34F2-ΔbclA spores. (A) Hydrophobicity measured with various volumes of hexadecane. (B) Hydrophobicity differences between 34F2 and 34F2-ΔbclA spores compared with a second hydrocarbon, toluene. (more ...)
Next, we assessed whether the greater hydrophobicity of wild-type versus bclA
mutant spores would still be evident in the presence of heat. We asked this question because Howell et al. (9
) previously reported that increased temperatures can alter the structure of macromolecules and expose internal hydrophobic moieties. Moreover, Doyle et al. suggested that increases in the hydrophobicity of spores because of heat treatment may result from the disruption of outer coat or exosporium proteins (7
). To examine the impact of heat on the relative hydrophobicity of the BclA-positive and -negative spores, we first had to test the effect of heat treatment alone on spore survival over time. No differences in the relative viabilities of wild-type and mutant spores after this heat treatment were noted (Fig. ). We then used a method described previously by Wiencek et al. (27
) to measure the effect of heat on hydrophobicity. The values obtained were compared with the values determined for the unheated controls, and the results are represented as the percent increase in hydrophobicity. As shown in Fig. , the mutant spores became strikingly more hydrophobic in the presence of heat than did wild-type spores even though heat did not differentially impact the viability of mutant and wild-type spores (Fig. ). One interpretation of the findings shown in Fig. is that spore proteins that are normally obscured by BclA are exposed in its absence and that these now-uncovered proteins are altered by heat treatment in a manner that leads to the increased hydrophobicity of mutant spores compared to that of wild-type spores.
FIG. 5. Evaluation of the impact of heat on hydrophobicity of 34F2 and 34F2-ΔbclA spores. (A) Effect of heat treatment on survival. (B) Percent increase in hydrophobicity compared to unheated controls. Both experiments were done in duplicate, and averages (more ...)
Further insight into how BclA affects the B. anthracis
spore surface is important for at least two reasons. First, such information may lead to a better understanding of the initial interaction of the spore with its host. Indeed, our data on a shorter MTD of 34F2-ΔbclA
versus 34F2 at a challenge dose of 106
spores, taken with a lower, albeit not statistically significantly different, LD50
of the BclA mutant than that of the wild-type strain for A/J mice, hint that BclA may slow spore germination in vivo. The fact that others have not reported any impact of BclA on virulence of B. anthracis
) may reflect the subtlety of a BclA effect. If that postulate is correct, variations between our group and others in bacterial or mouse strains used for these assays may explain the apparent discordance in the in vivo results. A second reason for further analyzing the influence of BclA on the B. anthracis
spore surface is the possibility that the hydrophobicity of BclA may dictate how spores initially interact with inanimate surfaces. If this theory proves to be the case, then an evaluation of chemical ways to interfere with or modify BclA may lead to more efficient methods of surface sanitation in cases of an inadvertent or deliberate release of spores as well as sterilization of equipment in the laboratory environment.