Sialylated Receptor Setting Influences Mycoplasma pneumoniae Attachment and Gliding Motility

Abstract

Mycoplasma pneumoniae is a common cause of human respiratory tract infections, including bronchitis and atypical pneumonia. M. pneumoniae binds glycoprotein receptors having terminal sialic acid residues via the P1 adhesin protein. Here, we explored the impact of sialic acid presentation on M. pneumoniae adherence and gliding on surfaces coated with sialylated glycoproteins, or chemically functionalized with α-2,3- and α-2,6-sialyllactose ligated individually or in combination to a polymer scaffold in precisely controlled densities. In both models, gliding required a higher receptor density threshold than adherence, and receptor density influenced gliding frequency but not gliding speed. However, very high densities of α-2,3-sialyllactose actually reduced gliding frequency over peak levels observed at lower densities. Both α-2,3- and α-2,6-sialyllactose supported M. pneumoniae adherence, but gliding was only observed on the former. Finally, gliding on α-2,3-sialyllactose was inhibited on surfaces also conjugated with α-2,6-sialyllactose, suggesting that both moieties bind P1 despite the inability of the latter to support gliding. Our results indicate that the nature and density of host receptor moieties profoundly influences M. pneumoniae gliding, which could affect pathogenesis and infection outcome. Furthermore, precise functionalization of polymer scaffolds shows great promise for further analysis of sialic acid presentation and M. pneumoniae adherence and gliding.

Fig. 1

M. pneumoniae attachment to chamber slides coated with laminin (A) or hCG (B) at the indicated levels. Each bar represents the mean and positive standard error of the mean for total cell counts for three separate experiments. SP4, data for chamber slides coated with serum glycoproteins in SP4 growth medium, which served as a positive control. * P < 0.001 for 2.0, 5.0, 10, and 20 μg laminin relative to the 0 μg negative control; ** P < 0.001 for 5.0, 10, 20, and 50 μg hCG relative to the 0 μg negative control; *** P < 0.001 for 50 μg hCG relative to the SP4 control.

Fig. 2

M. pneumoniae attachment to chamber slides coated with laminin, with and without pre-treatment with neuraminidase. Each bar represents the mean and positive standard error of the mean for attached cells for three separate experiments. SP4, data for chamber slides coated with serum glycoproteins in SP4 growth medium, which served as a positive control. + / −, with or without neuraminidase pre-treatment (P < 0.001).

Fig. 3

Gliding frequency for M. pneumoniae cells bound to chamber slides coated with laminin at the indicated amounts. Each bar represents the mean and positive standard error of the mean for gliding frequency for three separate experiments. SP4, data for chamber slides coated with serum glycoproteins in SP4 growth medium, which served as a positive control. * P<0.001 for 2.0, 5.0, 10, and 20 μg laminin relative to lower laminin levels; ** P < 0.001 for 20 μg laminin relative to the SP4 control.

Fig. 4

Schematic representation of poly(PFPA) brushes (blue) conjugated to glass (A), with the subsequent conjugation of hydrazine at various ratios with competing ethanolamine (B) dictating the density of hydrazide available for (C) ligation with the reducing end of sialyllactose (red).

Fig. 5

M. pneumoniae attachment to slides chemically functionalized with α-2,3- or α-2,6-sialyllactose, as indicated. Each bar represents the mean and positive standard error of the mean for total cell counts at a given sialyllactose percentage for three separate experiments. SP4, chamber slides coated with SP4 growth medium as a positive control. * P < 0.001 for cell numbers on 1.6% α-2,3-sialyllactose relative to the higher α-2,3-sialyllactose percentages; ** P < 0.001 for cell numbers on 32% and 80% α-2,6-sialyllactose relative to the lower α-2,6-sialyllactose percentages; *** P < 0.001 for cell numbers on SP4 relative to all percentages of α-2,6-sialyllactose.

Fig. 6

M. pneumoniae attachment to slides functionalized with sialyllactose with and without pre-treatment with neuraminidase. Each bar represents the mean and positive standard error of the mean for attached cells for three separate experiments. SP4, data for chamber slides coated with serum glycoproteins in SP4 growth medium, which served as a positive control. + / −, with or without neuraminidase pre-treatment (P < 0.001).

Fig. 7

Gliding frequency for M. pneumoniae cells attached to slides chemically functionalized with α-2,3-sialyllactose. Each bar represents the mean and positive standard error of the mean for gliding frequency for three separate experiments. SP4, data for chamber slides coated with serum glycoproteins in SP4 growth medium, which served as a positive control. *P < 0.001 for the indicated sialyllactose densities.

Fig. 8

Gliding tracks for M. pneumoniae attached to slides chemically functionalized with α-2,3- and α-2,6-sialyllactose at different ratios. (A) 1:0 α-2,3-sialyllactose and α-2,6-sialyllactose; (B) 1:1 α-2,3- and α-2,6-sialyllactose; (C) 1:2 α-2,3- and α-2,6-sialyllactose; and (D) 1:5 α-2,3- and α-2,6-sialyllactose. Gliding tracks were recorded at 1 frame/sec for 20-30 sec.