The physicochemical fingerprint of Necator americanus

Necator americanus, a haematophagous hookworm parasite, infects ~10% of the world’s population and is considered to be a significant public health risk. Its lifecycle has distinct stages, permitting its successful transit from the skin via the lungs (L3) to the intestinal tract (L4 maturing to adult). It has been hypothesised that the L3 larval sheath, which is shed during percutaneous infection (exsheathment), diverts the immune system to allow successful infection and reinfection in endemic areas. However, the physicochemical properties of the L3 larval cuticle and sheath, which are in direct contact with the skin and its immune defences, are unknown. In the present study, we controlled exsheathment, to characterise the sheath and underlying cuticle surfaces in situ, using atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). AFM revealed previously unseen surface area enhancing nano-annuli exclusive to the sheath surface and confirmed greater adhesion forces exist between cationic surfaces and the sheath, when compared to the emergent L3 cuticle. Furthermore, ToF-SIMS elucidated different chemistries between the surfaces of the cuticle and sheath which could be of biological significance. For example, the phosphatidylglycerol rich cuticle surface may support the onward migration of a lubricated infective stage, while the anionic and potentially immunologically active heparan sulphate rich deposited sheath could result in the diversion of immune defences to an inanimate antigenic nidus. We propose that our initial studies into the surface analysis of this hookworm provides a timely insight into the physicochemical properties of a globally important human pathogen at its infective stage and anticipate that the development and application of this analytical methodology will support translation of these findings into a biological context.


Materials and Methods
Water contact angle WCA was used to confirm the presence or absence of polymer coat. WC measurements were determined using a CAM 200 optical contact angle meter (KSV instruments Ltd) equipped with Pendant Drop Surface Tension Software (Version 3.42). Poly-L-lysine treated and untreated glass slides returned WCA's of 67.27 ± 5.33° ( Figure S1A) and 5.15 ± 1.09° ( Figure S1B), respectively (n=3).

Exsheathment Efficiency
The efficiency of larval exsheathment was determined by depositing ensheathed larva (n=20) suspended in deionised water on the surface of either poly-L-lysine coated and uncoated glass surfaces (n=3), which were incubated at 37 °C for 30 minutes. The number of exsheathed larva, ensheathed larva and sheaths were counted after incubation from which a percentage of exsheathment was calculated ( Figure S1C).

Atomic Force Microscopy
The nano-annuli provide the sheath with an enhanced surface area, when compared to the L3 cuticle. The surface area of the sheath can be approximated through calculation of the surface area for a single nano-annulus, using the elliptical cylinder surface area approximation equation.

Principal component analysis (PCA)
Primarily, PCA of the complete dataset (12 different larva & their corresponding 12 sheaths) was applied to determine if the surfaces are statistically different and can be differentiated from each other. Preliminary analysis of the normalised eigenvalues on the first ten principal components shows PC 1 (59.6 %), 2 (18.9 %) and 3 (12.0 %) account for 90.5 % of the variability within the data set Figure S4. In depth analysis of the principal component scores confirms the complexity of the biological surfaces under investigation as no component alone separate the L3 cuticle and sheath surfaces in the vertical axis, Figure S5 A-C.  Figure S5 D-E. Specifically, the component pair of PC2 & PC3 exhibits the greatest separation. When a three dimensional scores plot is generated for the first three principal components the data unambiguously separates into two distinct populations.
Secondly, through detailed analysis of the PCA loadings, calculation of the significant differences between the L3 cuticle and sheath, application of data filters and image analysis mass ions that are unique or were significantly expressed on each surface were identified. Through examination of both mean centred and auto-scaled loadings for the first three principal components it was established PC2 and PC3 correspond to markers for the sheath and L3 cuticle, Figure S6. Specifically, the sheath could be identified by low and high loadings on PC2 and PC3, respectively, whereas the L3 cuticle could be identified by high and low loadings on PC2 and PC3, respectively. However, due to the complexity of the dataset under consideration 95% confidence limits on PC loadings alone were not able to identify mass ions that were significantly expressed on L3 cuticle or sheath surface.
MCR analysis -negative polarity Images for highly loaded mass ions for MCR component 1-4 are shown in Figures S7-10.
Glass Substrate -Analysis of the highly loaded mass ions on MCR component 3 (glass substrate) indicate the presence of elemental ions, such as O -(16.00 u) and Cl -(34.97 u), as well as low mass carbon functional groups including CN-(26.00 u), CNO -(42.00 u) and C2H -(25.01). Additionally, phosphate related secondary ions such as PO2 -(62.97 u) and PO3 -(78.96 u) and silicate ion SiHO3 -(79.97 u) are also observed, which can be anticipated from this surface. The elemental ions, phosphates and silicate are located in greater abundance on the glass substrate, whereas the low molecular weight carbon functional groups are distributed throughout the field of view and may be residues of poly-L-lysine coating ( Figure S9).
Poly-L-lysine -The highly loaded carbon nitrogen compounds identified as the CNO -(42.00 u), CN -(26.00 u) and C2H2N -(40.02 u) secondary ions are distributed throughout the field of view for the MCR component 4 ( Figure S10). These are common mass fragments for poly-L-lysine and it has coated the glass substrate as well as the L3 cuticle and sheath. Figure S11 shows MCR residuals scores image and corresponding MCR Component loadings and mass assignments.
MCR analysis -positive polarity Preliminary screening of the hyperspectral data set with PCA indicated five components that account for distinct variability within the dataset. Based on these findings MCR analysis was conducted using five components. Figure S12 shows false colour heat maps and residuals for highly loaded mass ions for the positive polarity and show MCR 1 & 4 are specific for emerging L3 cuticle and sheath respectively, whereas MCR components 2, 3 and 5 demonstrate non-specificity to observed surfaces.
Analysis of the loadings plots for the highly loaded mass ions for MCR component 1 Figure S13A reveals the presence of a high degree of CxHxOx (Table S2) chemistry on the surface of the cuticle, of which CH5O + (33.04 u), C2H3O2 + (59.02 u), C2H4O2 + , C3H7O2+ (75.05 u), demonstrate specificity, Figure S14. Although MCR component 4 demonstrate specificity for the sheath, when further analysis is conducted on the highly loaded ions ( Figure S13B) none highly loaded compounds are specific for the sheath, Figure S15. This observation was confirmed using the statistical scoring which showed no positive ions are unique of significantly found on the surface of the sheath.

Further statistical analysis
Correlation coefficient analysis and statistical scoring were used to confirm the findings from MCR analysis and to show L3 cuticle and sheath are chemical different entities. It is important to note these methods utilized data from 12 different partially exsheathed N. americanus, for which 24 different regions of interest were defined (12 L3 cuticles and 12 sheaths), whereas MCR analysis was conducted on a hyperspectral dataset for entire fields of view containing a mixture of partially exsheathed helminths, glass substrate and poly-L-lysine.
Correlation coefficient analysis -Calculation of the correlation coefficients between two variables, the N. americanus L3 cuticle and sheath, permits interpretation of their linear interdependence, based on the coefficients sign (±) and strength (-1< correlation coefficient >1). As expected, all coefficients calculated for this data set are positive. This is because the data collected describes the presence and count for mass ions from a peak list rather than an absolute decrease from predetermined quantities. However, analysis of the strength of the correlation between N. americanus L3 cuticle and sheath surfaces indicates there are differences between their corresponding normalised m/z ions counts, Figure S16. This is implied in regions comparing the L3 cuticle and sheath, where the average correlation coefficient is 0.85 ± 0.09, whereas regions comparing L3 cuticle and L3 cuticle or sheath and sheath are 0.90 ±0.08 and 0.87 ± 0.12, respectively. Further analysis of the data-set suggest the correlation coefficients for the positive data set comparing L3 cuticle and sheath positive ions are weak and could be the source of chemical differences,. It is important to note when there are subtle differences in the correlation coefficients, as described in this example, it is challenging to isolate sources of variance. Bearing this in mind MCR (main article) and PCA analysis (below) was applied to data set to isolate components that have the strongest anti-correlation or variance.
Statistical Scoring -To refine the data set further, masses that were significantly expressed on either L3 cuticle or sheath surfaces were identified by combining data filters with Student's t-test. Data filters were applied to highlight masses with a minimum of twofold greater abundance on either the L3 cuticle or sheath, whilst Student's t-test was performed to confirm the statistical significance (p<0.01) Based on the data processing described above 60 (7.3% of all masses surveyed) and 21 (2.6% of all masses surveyed) mass ions were recorded to be unique or were significantly expressed on the L3 cuticle and sheath, respectively (Table S3).
Normalised mass intensities shows representative mass spectra for mass ions for the L3 cuticle (152.98 u, 33.04 u, 75.05 u Figure               Other Supporting Information for this manuscript includes the following: S1 Movie. Exsheathment behaviour of N. americanus at 37 °C in the absence of poly-L-lysine coated glass substrate.

S2
Movie. Exsheathment behaviour of N. americanus at 20 °C in the absence of poly-L-lysine coated glass substrate.

S3
Movie. Exsheathment behaviour of N. americanus at 20 °C in the presence of poly-L-lysine coated glass substrate. This video was captured using an inverted optical microscope and shows a mobile larva on top of stationary larva. The mobile larva is able to move the centre of its anatomy, because it is only in partial in contact with the poly-L-lysine surfaces at the head and tail. Whereas full the length of stationary larva is in contact with the coated surface and as a result its movement is restricted.

S4
Movie. Exsheathment behaviour of N. americanus at 37 °C in the presence of poly-L-lysine coated glass substrate (single larval exsheathment).