An ortholog of the Leptospira interrogans lipoprotein LipL32 aids in the colonization of Pseudoalteromonas tunicata to host surfaces

Abstract

The bacterium Pseudoalteromonas tunicata is a common surface colonizer of marine eukaryotes, including the macroalga Ulva australis.Genomic analysis of P. tunicata identified genes potentially involved in surface colonization, including genes with homology to bacterial virulence factors that mediate attachment. Of particular interest is the presence of a gene, designated ptlL32, encoding an ortholog to the Leptospira lipoprotein LipL32, which has been shown to facilitate the interaction of Leptospira sp. with host extracellular matrix (ECM) structures and is thought to be an important virulence trait for pathogenic Leptospira. To investigate the role of PtlL32 in the colonization by P. tunicata we constructed and characterized a ΔptlL32 mutant strain. Whilst P. tunicata ΔptlL32 bound to an abiotic surface with the same capacity as the wild type strain, it had a marked effect on the ability of P. tunicata to bind to ECM, suggesting a specific role in attachment to biological surfaces. Loss of PtlL32 also significantly reduced the capacity for P. tunciata to colonize the host algal surface demonstrating a clear role for this protein as a host-colonization factor. PtlL32 appears to have a patchy distribution across specific groups of environmental bacteria and phylogenetic analysis of PtlL32 orthologous proteins from non-Leptospira species suggests it may have been acquired via horizontal gene transfer between distantly related lineages. This study provides the first evidence for an attachment function for a LipL32-like protein outside the Leptospira and thereby contributes to the understanding of host colonization in ecologically distinct bacterial species.

Keywords: LipL32; Pseudoalteromonas; algae; bacterial attachment; host-microbe interaction; marine bacteria; seaweed/s.

Figure 1

Attachment of P. tunicata WT (solid fill) and ΔptlL32 (vertical stripes) to a polystyrene well plate after 6 h of incubation. The numbers of cells attached per mm³ were determined using direct counts of detached bacteria in a Helber bacterial counting chamber. Significance was assessed using the Students unpaired, two-tailed t-test (p > 0.8). Error bars represent standard deviation.

Figure 2

Attachment to Matrigel™ after 2 h (A) and 6 h (B) for ΔptlL32 (horizontal stripes) compared to WT (solid fill) and C ΔptlL32 (small squares). The number of cells attached per mm³ for each strain was estimated using direct counts of detached bacteria in a Helber bacterial counting chamber. Averages are shown as columns with n = 9. Error bars represent standard deviation. ^*Indicates significance with a p < 0.001 using an ANOVA.

Figure 3

Representative confocal laser scanning microscopy images of GFP-labeled WT (A) and GFP-labeled ΔptlL32 (B) attached to the surface of U. australis. Green fluorescent cells were enumerated using ImageJ software. Images were captured using a FV1000 confocal laser-scanning microscope. Scale bar represents 20 μm.

Figure 4

Average number of cells attached to (A), and cell aggregates (defined as a tight cluster of >10 cells) (B) on U. australis for GFP-labeled WT (solid fill) and GFP-labeled ΔptlL32 (horizontal stripes) after 6 h incubation. The number of green fluorescing cells or aggregates in each field of view was counted using ImageJ and an average expressed for cells per mm². Error bars represent standard deviation. Error bars represent standard deviation at 45 replicates and ^* denotes a significant difference with a p < 0.001 using the unpaired Students two-tailed t-test.

Figure 5

Maximum-likelihood tree of PtlL32 and orthologous protein sequences in non-Leptospira species. Five representative Leptospira LipL32 sequences were included for comparison with the non-Leptospira sequence. NCBI GenBank accession numbers for each protein sequence is provided in brackets. Bootstrap values for 100 replicates are shown for each node. The scale bar represents 10% sequence divergence.