[PMC free article] [PubMed] [CrossRef] [Google Scholar] 57
[PMC free article] [PubMed] [CrossRef] [Google Scholar] 57. in children who have significant comorbidities (8, 9). Invasive NTHi infections are fatal in 10% of children between 2 and 4?years of age and in 17% of children under the age of 1 1 (10, 11). The increase in invasive disease caused by NTHi is likely due to many factors, including increased numbers of vulnerable patient populations, rather than being solely due to Hib vaccine-induced strain alternative (6). Sialic acids are a diverse group of carboxylated nine carbon-backbone sugars (12, 13) with genes (Fig.?1) (45). Open in a separate windows FIG?1 Overview of the sialic acid catabolic and LOS biosynthesis pathways of NTHi for sialic acids Neu5Ac (purple) c-Fms-IN-8 and Neu5Gc (blue). Depending on its needs, NTHi can negate toxic building up of sialic acid by funneling extra sugar through the catabolic pathway or can convert sialic acid to an activated state for LOS sialylation. Around the inner membrane (IM), the biosynthesis pathway converts sialic acid to CMP-sialic acid, which acts as an electron donor to drive the transfer of sialic acids as terminal sugars to LOS. The sialylated LOS is usually flipped through the periplasmic space (PS) onto the outer membrane (OM). In the present study, we examined the uptake and presentation of Neu5Gc and Neu5Ac around the NTHi cell surface to better understand the mechanism of NTHi induction of anti-Neu5Gc antibodies in humans. RESULTS Neu5Gc is usually efficiently utilized as a carbon source. The utilization of Neu5Ac and its metabolic fate have been characterized in NTHi previously (46), but a study of the utilization of Neu5Gc by NTHi has not yet been carried out, although NTHi has been proposed to be a key antigen in the generation of anti-Neu5Gc antibodies (39). It was proposed that generation of these anti-Neu5Gc antibodies against NTHi requires incorporation of Neu5Gc from human serum acquired from the diet into the LOS by NTHi (39), likely through promiscuity of the LOS biosynthesis machinery or catabolic pathway (Fig.?1). c-Fms-IN-8 We hypothesized that there may be a bias in sialic acid utilization by NTHi that drives incorporation of the relatively scarce Neu5Gc into NTHi LOS. For example, in cases of inefficient catabolism of Neu5Gc as a carbon source c-Fms-IN-8 (relative to Neu5Ac), the lower rate of flux may generate a pool of Neu5Gc that is available for activation by addition of CMP and may thereby drive its preferential incorporation into LOS (Fig.?1). In fact, growth of NTHi strain 2019 using sialic acid-free chemically defined RPMI 1640 medium supplemented with 1% (vol/vol) hemin and 20?g/ml NAD (sRPMI medium) (47) and supplemented with either Neu5Ac or Neu5Gc as the sole carbon source revealed c-Fms-IN-8 that there was no difference in the growth rates of NTHi on these distinct sialic acids (Fig.?2). Open in a separate windows FIG?2 Growth of NTHi strain 2019 Rabbit Polyclonal to ABHD12 in sialic acid-free RPMI media. The medium was supplemented with a single carbon source, and bacterial growth was monitored for 12?h. The growth curves of Neu5Ac and Neu5Gc were found to be nearly identical and are superimposed. Preferential addition of Neu5Ac or Neu5Gc on NTHi LOS. On the basis of the results presented in Fig.?2, we concluded that Neu5Ac and Neu5Gc are utilized equally well by NTHi as a sole carbon source, and previous c-Fms-IN-8 work demonstrated that this sialic acid transporter SiaP bound Neu5Ac and Neu5Gc with comparable affinities (48). This indicated that this transport and catabolic pathways were unbiased in the utilization of these two sialic acids (Fig.?1). To investigate the potential for bias in the incorporation of Neu5Ac or Neu5Gc in the LOS biosynthesis pathway, NTHi strain 2019 was produced on sialic acid-free sRPMI media supplemented with various molar ratios of Neu5Ac and Neu5Gc (Fig.?3). NTHi LOS was purified from these cultures, and the amount of Neu5Ac and Neu5Gc present on LOS was determined by sugar analysis using.