AFM has become an important tool for characterizing the physical properties of bacterial surface molecules, including LPS, proteins, extracellular polymeric substances, and flagella (5
). In some cases, the force of adhesion between a bacterium and an AFM tip has been shown to correlate with LPS length, such as we observed on several strains of E
interacting with a silicon nitride AFM probe (39
). For P
in the present study, we did not see a direct correlation with LPS length and forces of adhesion to silicon nitride (see Fig. S3A in the supplemental material). However, there are important differences between the E
strains used previously and the P
strains used in the present study. In general, P
LPS cores have been found to be uncapped in about 80% of the cases (12
), while for E
, only ~10% of LPS molecules are uncapped (21
was also only compared across strains, such as by comparing E
O157:H7 (which has long LPS) to E
ML35 (with no O antigen and thus very short LPS). For different E
strains, we generally observed that the ones with longer LPS exhibited higher forces of adhesion to silicon nitride. For the P
mutants studied here, comparison of isogenic strains revealed more subtle differences which can be attributed specifically to the presence and/or presentation of LPS on the bacterial cell surface and not to differences in LPS composition.
Western blotting suggests that A-band LPS is longer than most of the O antigen in the DΔM strain (see Fig. S4 in the supplemental material). The A band would therefore mask the LPS of the DΔM strain, as well as the LPS core plus one O-antigen unit of the Wzy::GM mutant. Our AFM experiments and steric modeling results confirm that the top LPS layer on the Wzy::GM and DΔM mutants is indeed composed of the same type of molecules. Both mutants showed very similar LPS length and compressibility parameters which were also not significantly different. The decreased spacing in DΔM is consistent with the expected presence of underlying B-band LPS molecules. Thus, we conclude that the longest LPS in the Wzy::GM and DΔM strains is likely composed of A-band molecules.
The wild-type strain exhibited the broadest distributions of both LPS length and adhesion forces. Any of the introduced mutations narrowed the adhesion force distribution (Fig. ). The LPS length distribution only became very narrow when nearly all of the LPS was removed, as was the case for the Wzy::GM mutant, or when the O-antigen chain length preference was lost, as in the case of DΔM (Fig. ). This can be attributed to the presence of A-band LPS, which covers the B-band molecules on the surface of these two mutants. Overall, the wild type was the most adhesive to silicon nitride and adhesion decreased with any mutations introduced. This suggests that the wild-type strain is already optimized to have the maximum adhesion in nature.
Although LPS length and adhesion force were not well correlated with one another, some of the other physical properties showed interdependence. LPS length showed a positive correlation with delta offset (R2 = 0.65) in that δ increased as Lo increased (see Fig. S3C in the supplemental material). This is the result of the AFM tip making a stiff contact further away from the cell membrane with an increase in the thickness of the LPS layer. LPS length and spacing showed a weak correlation for the O-antigen-expressing strains (see Fig. S3B in the supplemental material). The reason for this interdependence is not clear.
The relative values of the parameters of the AdG model were consistent with previous biological findings and expectations. The strongest agreement was observed in the Lo parameter of the fitted equation. The median LPS lengths of the wild type and ΔWzz1 showed no statistically significant differences, as the AFM tip interacts only with the surface of the LPS layer, which in both cases is built up of the very long B-band LPS. Still, ΔWzz1 showed slightly longer LPS molecules, because removing the long O-antigen side chain would result in an excess amount of saccharide O-antigen subunits. The double mutant and Wzy::GM were also not significantly different from each other, as in this case, the A-band chains mask the underlying B-band molecules and thus form the topmost LPS layer, which we measured to have a length of about 36 nm.
The δ offset parameter of the AdG equation is an experimental variable and is dependent on both the cantilever stiffness and the tip radius. Assuming that these specifications are similar for the different AFM probes that we used, the δ offset can provide insight into the LPS layer's physical properties. The median δ offset values for the wild type, ΔWzz1, and ΔWzz2 showed no significant difference from each other, indicating that the O-antigen part of the LPS molecule has uniform compressibility, which is independent of the synthesis mechanism. The median values for DΔM and Wzy::GM were also not statistically significantly different, suggesting that the A-band LPS molecules have the same compressibility throughout the different mutants as well.
Bacteria, like most other microbes and cells, are negatively charged. The magnitude of the charge, however, is dependent upon the bacterial surface structures and is related to the cell's ability to attach to various substrates (44
LPS varies among the different serogroups, and its chemical structure affects the bacterial surface charge. P
PA103 LPS core consists mainly of neutral sugars but has several negatively charged sites such as 3-deoxy-d
-octulosonic acid residues and phosphate groups. The A band, composed of d
-rhamnose trisaccharide repeating units, and the B-band LPS, consisting of trisaccharide units of one glucose and two N
-acetylfucosamine residues, are electroneutral (28
). Our zeta potential measurements are consistent with the expected charge distribution on the P
surface. The effect of the negatively charged LPS core molecules is greatest for the Wzy::GM mutant due to its lack of O antigen (zeta potential of −45.0 ± 0.5 mV). These charges are then masked by the addition of O-antigen repeating units for the wild type and the other three mutants, as indicated by the increase in zeta potential. Studies aimed at exploring the effect of LPS mutation on attachment to various biotic and abiotic surfaces could further elucidate the role of LPS structure in bacterial adhesion.
Relating LPS physical properties and adhesion force to bacterial pathogenicity can provide a convenient in vitro
way to discriminate between virulent and avirulent strains. The silicon nitride AFM probe is not truly representative of the complex pathogen-host interactions, but it has been successfully used as a model surface. Previous work has found correlations between LPS length and adhesion force (39
) and between adhesion force and virulence (34
) in different strains of the same species using a silicon nitride cantilever. In this work, we compared isogenic LPS mutants and did not find a correlation between LPS length and virulence, but we noted a relationship between adhesion force and virulence. Wzz deletion mutants lacking the preferences for wild-type lengths of the O-antigen side chain were tested in a mouse acute pneumonia model of infection. At 7 × 105
CFU, mice infected with the ΔWzz2 mutant did not show any significant difference from mice infected with wild-type PA103, while mice infected with the ΔWzz1 mutant exhibited much longer survival times. This shows that LPS length cannot be reliably used as a predictor of virulence, because the biological differences among the mutants overrule the effect of variations in LPS layer physical conformation. At a lower dose averaging 2 × 105
CFU, the ΔWzz2 mutant exhibited a slight attenuation compared to the wild type, although statistical tests could not determine a significant difference due to the obstacles in testing a larger number of mice. These results suggest that adhesion force can be used as a predictor of virulence at low doses, which in fact more accurately represent natural infection settings. The wild-type PA103 proved to be the most virulent and also showed the highest adhesion forces, while ΔWzz1 and ΔWzz2 showed lower force profiles and, respectively, attenuated and slightly attenuated pathogenicity. We speculate that the difference between the wild-type and ΔWzz2 strains will be even more pronounced at lower infection doses. At 2 × 105
CFU, the double deletion mutant proved to be completely avirulent, suggesting an additive effect of the removal of both long and very long O-antigen side chains. Other studies have demonstrated that LPS rough mutants exhibit much higher 50% lethal doses than the respective wild-type organisms (11
). No comparison of adhesion force magnitude can be made between the O-antigen-expressing strains (wild-type PA103 and the ΔWzz1 and ΔWzz2 mutants) and the mutants in which A-band LPSs make up the topmost part of the LPS layer (Wzy::GM and DΔM), as the interaction forces in the two groups arise from contact with chemically different molecules. We can, however, predict, based on the adhesion force profiles of the two mutants, that Wzy::GM will be more attenuated than DΔM, although it will be difficult to determine if subtle differences exist, given the avirulence of the DΔM mutant.
The deletion mutants created in this study exhibit a more pronounced phenotype than the insertional mutants created previously (26
) with respect to the decreased presentation of O-antigen chains of particular lengths, which facilitated AFM experiments and modeling and might have also led to a further reduction in virulence. However, when the deletion mutants generated here were tested in the acute pneumonia model at the same dose as that previously used (26
), very similar survival curves were obtained. This indicates that the insertional mutants already altered the presentation of O-antigen chain length enough that virulence was decreased. Further attenuation was not detected, even when there was a complete lack of O antigen of these particular lengths.